Optical imaging system using lateral illumination for digital assays

ABSTRACT

A compact optical imaging system including a single filter and a light source that provides lateral illumination for bead detection in digital assays. The light source is configured to emit light toward the detection vessel. The single filter is positioned to receive light reflected from a sample in the detection vessel, that originated from the light source, and receive an output from a sample in the detection vessel. A detector is configured to receive a portion of the reflected light and a portion of the output that passes through the single filter.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a non-provisional of and claims benefit of U.S.Provisional Patent Application No. 62/437,534, filed on Dec. 21, 2016,the entire contents of which are incorporated herein by reference.

TECHNICAL FIELD

This disclosure relates to an optical imaging system including a singlefilter and a light source that provides lateral illumination for analyteanalysis in digital assays.

BACKGROUND OF THE INVENTION

Devices and methods that can accurately analyze analyte(s) of interestin a sample are essential for diagnostics, such as for examplediagnosing a disease, disorder or condition, prognostics, environmentalassessment, food safety, detection of chemical or biological warfareagents and the like. Most current techniques for quantifying low levelsof analyte molecules in a sample use amplification procedures toincrease the number of reporter molecules to provide a measurablesignal. Examples of current techniques include enzyme-linkedimmunosorbent assays (ELISA) for amplifying the signal in antibody-basedassays, as well as the polymerase chain reaction (PCR) for amplifyingtarget DNA strands in DNA-based assays. Most detection schemes requirethe presence of a large number of molecules in the ensemble for theaggregate signal to be above the detection threshold. This requirementlimits the sensitivity of most detection techniques and the dynamicrange (i.e., the range of concentrations that can be detected). Many ofthe known methods and techniques are further plagued with problems ofnon-specific binding, which leads to an increase in the backgroundsignal and limits the lowest concentration that may be accurately orreproducibly detected.

Digital ELISA is a candidate for next generation of immunoassay as itcan detect one molecule of enzyme using a conjugate. See FIGS. 1 and 2.In digital ELISA, target molecules are captured on beads between acapture antibody and a detection antibody, wherein the detectionantibody is bound to an enzyme. The beads are then entrapped in adroplet chamber with the substrate of the enzyme, and the aqueous phaseis displaced by a heavy oil, allowing removal of the aqueous phasebefore analysis.

In bead-based digital ELISA, single beads are encapsulated inmicrochambers of the array. Some of the chambers in which a bead hascaptured an immune-complex species provide bright spots uponfluorescence imaging (i.e., a bright chamber). The percentage ofchambers having beads present is correlated to concentration of antigen.Therefore, it is necessary to identify positions of beads and enzymes inthe microchambers of the array.

Optical imaging is a method to determine the location of beads in anassay. Optical imaging can also detect data for a large number (over10,000) of microchambers at a time using bead/enzyme dual channelimaging. However, conventional optical systems for dual channel imagingare large and expensive to mount on existing products because of thecomplicated optics for fluorescence/fluorescence type of bead/enzymedual channel imaging. Conventional optical systems require multipleoptical filters for the respective channels, and an external actuatorfor exchanging the modes.

SUMMARY OF THE INVENTION

Accordingly, it is desirable to provide a simplified optical imagingsystem for digital immunoassay that provides for a compact and low costdigital immunoassay device. Embodiments of the invention provide animaging system using light scattering optics for digital immunoassaywhere size and cost are substantially reduced compared to a conventionaloptical imaging system.

Embodiments of the invention simplify the optical imaging system byemploying a light source that applies light scattering to the assay forbead detection. The optical imaging system utilizeslight-scattering/fluorescence type and has been simplified by removingoptical components and the corresponding actuator structure for the modeexchange used in a conventional system. To demonstrate the power of theoptical imaging system according to embodiments of the system, a compactdevice for digital ELISA was designed and tested. The embodiments of theoptical imaging system demonstrated good performance and were comparableor superior to a conventional optical imaging system. (Noteworthyspecifications are summarized in FIG. 1.) The detector is approximatelyseveral 10s and 100 times smaller in volume and cost, respectively.

In one embodiment, the invention provides a compact digital assayapparatus comprising a detection vessel, a light source configured toemit light toward the detection vessel, a single filter and a detector.The single filter is positioned to receive light reflected from a samplein the detection vessel, that originated from the light source, andreceive an output from a sample in the detection vessel. The detector isconfigured to receive a portion of the reflected light and a portion ofthe output that passes through the single filter.

In another embodiment, the invention provides a compact digital assayapparatus comprising a detection vessel, a first light source configuredto emit light toward the detection vessel and at an angle relative tothe sample array, a second light source configured to emit light towardthe detection vessel, a single filter, and a detector. The single filteris positioned to receive light reflected from a sample in the detectionvessel, that originated from the first light source, and receive afluorescence output from a sample in the detection vessel. The detectoris configured to receive a portion of the reflected light and a portionof the fluorescence that passes through the single filter.

In a further embodiment, the invention provides a compact digital assayapparatus comprising a sample array having a plurality of wells, a firstlight source configured to emit light toward the sample array at anangle relative to the sample array to illuminate a sample in the samplearray, a second light source configured to emit light toward the samplearray without using a mirror or other reflective object, the secondlight source further configured to activate a sample in the sample arrayto emit an output, a filter, and a detector. The filter is positioned toreceive light reflected from the sample array that originated from thefirst light source, and receive the output from the sample in the samplearray. The detector is configured to receive the reflected light and theoutput from the sample that pass through the filter and to generateoptical data identifying which wells contain a bead and which wellscontain a label.

In yet another embodiment, the invention provides a compact digitalassay apparatus comprising a sample array including a plurality ofsamples positioned in a plurality of nanowells, a light sourceconfigured to emit light toward the sample array at an angle relative tothe sample array to illuminate a sample in the sample array, the lightsource having a wavelength between 450 nm and 550 nm, and a detectorconfigured to receive a portion of light reflected from the sample.

Other aspects of the invention will become apparent by consideration ofthe detailed description and accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The details of the subject matter set forth herein, both as to itsstructure and operation, may be apparent by study of the accompanyingfigures, in which like reference numerals refer to like parts. Thecomponents in the figures are not necessarily to scale, emphasis insteadbeing placed upon illustrating the principles of the subject matter.Moreover, all illustrations are intended to convey concepts, whererelative sizes, shapes and other detailed attributes may be illustratedschematically rather than literally or precisely.

FIG. 1 is a comparison of a simplified optical imaging system accordingto an embodiment of the present invention and a conventional opticalimaging system.

FIG. 2 is a perspective view of an optical imaging system according toan embodiment of the present invention.

FIG. 3 shows several views of the optical imaging system illustrated inFIG. 2.

FIG. 4 is a schematic of an optical imaging system according to anembodiment of the present invention.

FIG. 5 schematic illustration of a microchamber array for digitalimmunoassay and a mechanism of fluorescent signal amplification by anenzymatic reaction (top images); comparison of differences betweenoptical imaging system of a convention system and the optical imagingsystem illustrated in FIG. 2.

FIG. 6 illustrates images acquired by an optical imaging systemillustrated in FIG. 5.

FIG. 7 illustrates images acquired by an optical imaging systemillustrated in FIG. 5.

FIG. 8 illustrates images acquired by an optical imaging systemillustrated in FIG. 5.

FIG. 9 illustrates images acquired by an optical imaging systemillustrated in FIG. 5.

FIG. 10 illustrates images acquired by an optical imaging systemillustrated in FIG. 5.

FIG. 11 illustrates images acquired by an optical imaging systemillustrated in FIG. 5.

FIG. 12 illustrates images acquired by an optical imaging systemillustrated in FIG. 5.

DETAILED DESCRIPTION 1. DEFINITIONS

Before the embodiments of the present disclosure are described, it is tobe understood that this invention is not limited to particularembodiments described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

“Comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” andvariants thereof, as used herein, are intended to be open-endedtransitional phrases, terms, or words that do not preclude thepossibility of additional acts or structures. The singular forms “a,”“and” and “the” include plural references unless the context clearlydictates otherwise. The present disclosure also contemplates otherembodiments “comprising,” “consisting of” and “consisting essentiallyof,” the embodiments or elements presented herein, whether explicitlyset forth or not.

For the recitation of numeric ranges herein, each intervening numberthere between with the same degree of precision is explicitlycontemplated. For example, for the range of 6-9, the numbers 7 and 8 arecontemplated in addition to 6 and 9, and for the range 6.0-7.0, thenumber 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 areexplicitly contemplated.

“Affinity” and “binding affinity” as used interchangeably herein referto the tendency or strength of binding of the binding member to theanalyte. For example, the binding affinity may be represented by theequilibrium dissociation constant (KD), the dissociation rate (kd), orthe association rate (ka).

“Analog” as used herein refers to a molecule that has a similarstructure to a molecule of interest (e.g., nucleoside analog, nucleotideanalog, sugar phosphate analog, analyte analog, etc.). An analyte analogis a molecule that is structurally similar to an analyte but for whichthe binding member has a different affinity.

“Analyte”, “target analyte”, “analyte of interest” as usedinterchangeably herein, refer to an analyte being measured in themethods and devices disclosed herein. Analytes of interest are furtherdescribed herein.

“Antibody” and “antibodies” as used herein refers to monoclonalantibodies, multispecific antibodies, human antibodies, humanizedantibodies (fully or partially humanized), animal antibodies such as,but not limited to, a bird (for example, a duck or a goose), a shark, awhale, and a mammal, including a non-primate (for example, a cow, a pig,a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, aguinea pig, a cat, a dog, a rat, a mouse, etc.) or a non-human primate(for example, a monkey, a chimpanzee, etc.), recombinant antibodies,chimeric antibodies, single-chain Fvs (“scFv”), single chain antibodies,single domain antibodies, Fab fragments, F(ab′) fragments, F(ab′)2fragments, disulfide-linked Fvs (“sdFv”), and anti-idiotypic (“anti-Id”)antibodies, dual-domain antibodies, dual variable domain (DVD) or triplevariable domain (TVD) antibodies (dual-variable domain immunoglobulinsand methods for making them are described in Wu, C., et al., NatureBiotechnology, 25(11):1290-1297 (2007) and PCT International PatentApplication WO 2001/058956, the contents of each of which are hereinincorporated by reference), and functionally active epitope-bindingfragments of any of the above. In particular, antibodies includeimmunoglobulin molecules and immunologically active fragments ofimmunoglobulin molecules, namely, molecules that contain ananalyte-binding site. Immunoglobulin molecules can be of any type (forexample, IgG, IgE, IgM, IgD, IgA, and IgY), class (for example, IgG1,IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. For simplicity sake, anantibody against an analyte is frequently referred to herein as beingeither an “anti-analyte antibody” or merely an “analyte antibody.”

“Antibody fragment” as used herein refers to a portion of an intactantibody comprising the antigen-binding site or variable region. Theportion does not include the constant heavy chain domains (i.e., CH2,CH3, or CH4, depending on the antibody isotype) of the Fc region of theintact antibody. Examples of antibody fragments include, but are notlimited to, Fab fragments, Fab′ fragments, Fab′-SH fragments, F(ab′)2fragments, Fd fragments, Fv fragments, diabodies, single-chain Fv (scFv)molecules, single-chain polypeptides containing only one light chainvariable domain, single-chain polypeptides containing the three CDRs ofthe light-chain variable domain, single-chain polypeptides containingonly one heavy chain variable region, and single-chain polypeptidescontaining the three CDRs of the heavy chain variable region.

“Bead” and “particle” are used herein interchangeably and refer to asubstantially spherical solid support.

“Binding Protein” is used herein to refer to a monomeric or multimericprotein that binds to and forms a complex with a binding partner, suchas, for example, a polypeptide, an antigen, a chemical compound or othermolecule, or a substrate of any kind. A binding protein specificallybinds a binding partner. Binding proteins include antibodies, as well asantigen-binding fragments thereof and other various forms andderivatives thereof as are known in the art and described herein below,and other molecules comprising one or more antigen-binding domains thatbind to an antigen molecule or a particular site (epitope) on theantigen molecule. Accordingly, a binding protein includes, but is notlimited to, an antibody a tetrameric immunoglobulin, an IgG molecule, anIgG1 molecule, a monoclonal antibody, a chimeric antibody, a CDR-graftedantibody, a humanized antibody, an affinity matured antibody, andfragments of any such antibodies that retain the ability to bind to anantigen.

“Capture molecule” as used herein refers to a specific binding partneror specific binding member used to capture or immobilize an analyte ofinterest in a biological sample. A capture molecule is often onecomponent of a complex in addition to the analyte of interest and mayalso contain one or more detection molecules. The complex may optionallybe bound to a solid support.

“Component,” “components,” or “at least one component,” refer generallyto a capture antibody, a detection reagent or conjugate, a calibrator, acontrol, a sensitivity panel, a container, a buffer, a diluent, a salt,an enzyme, a co-factor for an enzyme, a detection reagent, apretreatment reagent/solution, a substrate (e.g., as a solution), a stopsolution, and the like that can be included in a kit for assay of a testsample, such as patient urine, serum, whole blood, tissue aspirate, orplasma sample, in accordance with the methods described herein and othermethods known in the art. Some components can be in solution orlyophilized for reconstitution for use in an assay.

“Contacting” and grammatical equivalents thereof as used herein refer toany type of combining action which brings a binding member intosufficiently close proximity with the analyte of interest in the samplesuch that a binding interaction will occur if the analyte of interestspecific for the binding member is present in the sample. Contacting maybe achieved in a variety of different ways, including combining thesample with a binding member, exposing a target analyte to a bindingmember by introducing the binding member in close proximity to theanalyte, and the like.

“Control” as used herein refers to a reference standard for an analytesuch as is known or accepted in the art, or determined empirically usingacceptable means such as are commonly employed. A “reference standard”is a standardized substance which is used as a measurement base for asimilar substance. For example, there are documented reference standardspublished in the U.S. Pharmacopeial Convention (USP-NF), Food ChemicalsCodex, and Dietary Supplements Compendium (all of which are available athttp://www.usp.org), and other well-known sources. Methods forstandardizing references are described in the literature. Alsowell-known are means for quantifying the amounts of analyte present byuse of a calibration curve for analyte or by comparison to an alternatereference standard. A standard curve can be generated using serialdilutions or solutions of known concentrations of analyte, by massspectroscopy, gravimetric methods, and by other techniques known in theart. Alternate reference standards that have been described in theliterature include standard addition (also known as the method ofstandard addition), or digital polymerase chain reaction.

“Detection molecule” as used herein refers to a specific binding partneror specific binding member that is used to detect the presence of and/orquantify or measure the amount of an analyte of interest in a biologicalsample. A detection molecule is often one component of a complex thatmay contain a one or more capture molecules and an analyte of interest.The complex may optionally be bound to a solid support.

“Detection” or “reaction” vessel as used herein refers a container orother apparatus that may contain a reaction mixture that may or may notcontain an analyte of interest. Examples of suitable “detection” or“reaction” vessels include a cuvette, a tube, the individual tube(s) ofthe tube plate, the hole(s) or well(s) in the microtiter plate, theindividual reaction well(s) (such as an array of wells such as amicrowell array or a nanowell array) or pit(s) in the test slide plateor assay array plate.

“Immobilized” as used herein, refers to a stable association of thefirst specific binding member with a surface of a solid support. By“stable association” is meant a physical association between twoentities in which the mean half-life of association is one day or more,e.g., under physiological conditions. In certain aspects, the physicalassociation between the two entities has a mean half-life of two days ormore, one week or more, one month or more, including six months or more,e.g., 1 year or more, in PBS at 4° C. According to certain embodiments,the stable association arises from a covalent bond between the twoentities, a non-covalent bond between the two entities (e.g., an ionicor metallic bond), or other forms of chemical attraction, such ashydrogen bonding, Van der Waals forces, and the like.

“Label” and “detectable label” as used herein refer to a moiety attachedto an antibody or an analyte to render the reaction between the antibodyand the analyte detectable, and the antibody or analyte so labeled isreferred to as “detectably labeled.” A label can produce a signal thatis detectable by visual or instrumental means. Various labels includesignal-producing substances, such as chromogens, fluorescent compounds,chemiluminescent compounds, radioactive compounds, and the like. Otherlabels are described herein. In this regard, the moiety, itself, may notbe detectable but may become detectable upon reaction with yet anothermoiety. Use of the term “detectably labeled” is intended to encompasssuch labeling.

“Monoclonal antibody” as used herein refers to an antibody obtained froma population of substantially homogeneous antibodies, i.e., theindividual antibodies comprising the population are identical except forpossible naturally occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigen. Furthermore, in contrast to polyclonalantibody preparations that typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Themonoclonal antibodies herein specifically include “chimeric” antibodiesin which a portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological.

“Polynucleotides” or “oligonucleotides” refer to nucleobase polymers oroligomers in which the nucleobases are connected by sugar phosphatelinkages (sugar-phosphate backbone). Exemplary poly- andoligonucleotides include polymers of 2′-deoxyribonucleotides (DNA) andpolymers of ribonucleotides (RNA). A polynucleotide may be composedentirely of ribonucleotides, entirely of 2′-deoxyribonucleotides orcombinations thereof. “Nucleic acid” encompasses “polynucleotide” and“oligonucleotides” and includes single stranded and double strandedpolymers of nucleotide monomers.

“Predetermined cutoff” and “predetermined level” as used herein refer toan assay cutoff value that is used to assess diagnostic, prognostic, ortherapeutic efficacy results by comparing the assay results against thepredetermined cutoff/level, where the predetermined cutoff/level alreadyhas been linked or associated with various clinical parameters (e.g.,presence of disease, stage of disease, severity of disease, progression,non-progression, or improvement of disease, etc.). The disclosureprovides exemplary predetermined levels. However, it is well-known thatcutoff values may vary depending on the nature of the immunoassay (e.g.,antibodies employed, reaction conditions, sample purity, etc.). Itfurther is well within the ordinary skill of one in the art to adapt thedisclosure herein for other immunoassays to obtain immunoassay-specificcutoff values for those other immunoassays based on the descriptionprovided by this disclosure. Whereas the precise value of thepredetermined cutoff/level may vary between assays, the correlations asdescribed herein should be generally applicable.

“Pretreatment reagent,” e.g., lysis, precipitation and/or solubilizationreagent, as used in a diagnostic assay as described herein is one thatlyses any cells and/or solubilizes any analyte that is/are present in atest sample. Pretreatment is not necessary for all samples, as describedfurther herein. Among other things, solubilizing the analyte entailsrelease of the analyte from any endogenous binding proteins present inthe sample. A pretreatment reagent may be homogeneous (not requiring aseparation step) or heterogeneous (requiring a separation step). Withuse of a heterogeneous pretreatment reagent, there is removal of anyprecipitated analyte binding proteins from the test sample prior toproceeding to the next step of the assay. The pretreatment reagentoptionally can comprise: (a) one or more solvents and salt, (b) one ormore solvents, salt and detergent, (c) detergent, (d) detergent andsalt, or (e) any reagent or combination of reagents appropriate for celllysis and/or solubilization of analyte.

“Quality control reagents” in the context of immunoassays and kitsdescribed herein, include, but are not limited to, calibrators,controls, and sensitivity panels. A “calibrator” or “standard” typicallyis used (e.g., one or more, such as a plurality) in order to establishcalibration (standard) curves for interpolation of the concentration ofan analyte, such as an antibody or an analyte. Alternatively, a singlecalibrator, which is near a predetermined positive/negative cutoff, canbe used. Multiple calibrators (i.e., more than one calibrator or avarying amount of calibrator(s)) can be used in conjunction to comprisea “sensitivity panel.”

“Receptor” as used herein refers to a protein-molecule that recognizesand responds to endogenous-chemical signals. When suchendogenous-chemical signals bind to a receptor, they cause some form ofcellular/tissue-response. Examples of receptors include, but are notlimited to, neural receptors, hormonal receptors, nutrient receptors,and cell surface receptors.

“Recombinant antibody” and “recombinant antibodies” refer to antibodiesprepared by one or more steps, including cloning nucleic acid sequencesencoding all or a part of one or more monoclonal antibodies into anappropriate expression vector by recombinant techniques and subsequentlyexpressing the antibody in an appropriate host cell. The terms include,but are not limited to, recombinantly produced monoclonal antibodies,chimeric antibodies, humanized antibodies (fully or partiallyhumanized), multi-specific or multi-valent structures formed fromantibody fragments, bifunctional antibodies, heteroconjugate Abs,DVD-Ig®s, and other antibodies as described in (i) herein.(Dual-variable domain immunoglobulins and methods for making them aredescribed in Wu, C., et al., Nature Biotechnology, 25:1290-1297 (2007)).The term “bifunctional antibody,” as used herein, refers to an antibodythat comprises a first arm having a specificity for one antigenic siteand a second arm having a specificity for a different antigenic site,i.e., the bifunctional antibodies have a dual specificity.

“Sample,” “test sample,” “biological sample,” “sample from a subject,”“fluid biological sample,” and “patient sample” as used herein may beused interchangeable and refer to fluid sample containing or suspectedof containing an analyte of interest.

As used herein “sample array” refers to a collection of one or more (ora plurality of) detection or reaction vessels.

As used herein, “signal generating compound” refers to any molecule,compound, protein or the like that can be converted to a detectableproduct or detectable label upon exposure to a suitable or appropriateconverting agent, such as a signal generating substrate. A “detectableproduct” or “detectable label” is any molecule, particle, or the like,that facilitates detection, by acting as the detected entity, using achosen technique known in the art. An example of a signal generatingcompound is an enzyme such as amylases, polynucleotidase, arginase,adenase, aminopolypeptidase, pepsin, lipases, catalase, tyrosinases,alcohol dehydrogenase, succinic dehydrogenase, diaphorase, glyoxalase,aldolase, glucose oxidase, horseradish peroxidase, a galactosidase (suchas beta-galactosidase), phosphatases, phosphorylases and hexokinases orcombinations thereof.

As used herein “signal generating substrate” refers any molecule,compound, protein, substance, particle, or the like, that can beconverted to or result in a signal generating compound being convertedto a detectable product or detectable label upon exposure to a suitableor appropriate converting agent, such as a signal generating compound. A“detectable compound” or “detectable label” is any molecule, particle,or the like, that facilitates detection, by acting as the detectedentity, using a chosen technique known in the art. Signal generatingsubstrates can be colorimetric, chemiluminescent or chemifluorescent. Anexample of a signal generating substrate is an enzymatic substrate, suchas a chemiluminescent substrate such as CDP-Star®, (disodium4-chloro-3-(methoxyspiro{1,2-dioxetane-3,2′-(5′-chloro)tricyclo[3.3.1.1.s-up.3,7]decane}-4-yl)phenylphosphate), CSPD®, or (disodium3-(4-methoxyspiro{1,2-dioxetane-3,2-(5′-chloro)tricyclo[3.3.1.1-.sup.3,7]decane}-4-yl)phenylphosphate); a luminescent substrate such as p-nitrophenyl phosphate,5-bromo-4-chloro-3-indolyl phosphate (BCIP), 4-nitro blue tetrazoliumchloride (NBT), or iodonitrotetrazolium (INT); a fluorescent substratesuch as 4-methylumbelliferyl phosphate (4-MUP); and a chromogenicsubstrate such as 5-bromo-4-chloro-3-indolyl phosphate (BCIP), disodium5-bromo-6-chloro-indolyl phosphate, or p-nitrophenyl phosphate.

“Specific binding partner” or “specific binding member” as usedinterchangeably herein refers to one of two or more different moleculesthat specifically recognize the other molecule compared to substantiallyless recognition of other molecules. The one of two different moleculeshas an area on the surface or in a cavity, which specifically binds toand is thereby defined as complementary with a particular spatial andpolar organization of the other molecule. The molecules may be membersof a specific binding pair. For example, a specific binding member mayinclude, but is not limited to, a protein, such as a receptor, anenzyme, and an antibody.

In addition to antigen and antibody specific binding pairs of commonimmunoassays, other specific binding pairs can include biotin and avidin(or streptavidin), carbohydrates and lectins, complementary nucleotidesequences, effector and receptor molecules, cofactors and enzymes,enzymes and enzyme inhibitors, and the like. Furthermore, specificbinding pairs can include members that are analogs of the originalspecific binding members, for example, an analyte-analog. Immunoreactivespecific binding members include antigens, antigen fragments, andantibodies, including monoclonal and polyclonal antibodies as well ascomplexes and fragments thereof, whether isolated or recombinantlyproduced.

“Solid support” refers to any material that is insoluble, or can be madeinsoluble by a subsequent reaction. The solid support can be chosen forits intrinsic ability to attract and immobilize a capture agent.Alternatively, the solid support can have affixed thereto a linkingagent that has the ability to attract and immobilize the capture agent.For example, the linking agent can include a charged substance that isoppositely charged with respect to the capture agent itself or to acharged substance conjugated to the capture agent. In general, thelinking agent can be any binding partner (preferably specific) that isimmobilized on (attached to) the solid support and that has the abilityto immobilize the capture agent through a binding reaction. The linkingagent enables the indirect binding of the capture agent to a solidsupport material before the performance of the assay or during theperformance of the assay. For examples, the solid support can beplastic, derivatized plastic, magnetic, or non-magnetic metal, glass orsilicon, including, for example, a test tube, microtiter well, sheet,bead, microparticle, chip, and other configurations known to those ofordinary skill in the art.

“Subject” and “patient” as used herein interchangeably refers to anyvertebrate, including, but not limited to, a mammal (e.g., cow, pig,camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat,dog, rat, and mouse, a non-human primate (for example, a monkey, such asa cynomolgous or rhesus monkey, chimpanzee, etc.) and a human). In someembodiments, the subject may be a human or a non-human. The subject orpatient may be undergoing other forms of treatment.

“Threshold” as used herein refers to an empirically determined andsubjective cutoff level above which acquired data is considered“signal,” and below which acquired data is considered “noise.” Acomputer program based on CUSUM (Cumulative Sums Algorithm) is employedto process acquired data and detect events based on threshold input fromthe user. Variation between users is avoided by detection of any manyevents as possible followed by filtering the data afterwards forspecific purposes. With a “loose” threshold a lesser number of eventswill be counted as signal. With a “tight” threshold a greater number ofevents will be counted as signal. Setting the threshold as loose ortight is a subjective choice based on the desired sensitivity orspecificity for an assay, and whether in a given assessment falsepositives or false negatives would be preferred.

“Treat”, “treating” or “treatment” are each used interchangeably hereinto describe reversing, alleviating, or inhibiting the progress of adisease, or one or more symptoms of such disease, to which such termapplies. Depending on the condition of the subject, the term also refersto preventing a disease, and includes preventing the onset of a disease,or preventing the symptoms associated with a disease. A treatment may beeither performed in an acute or chronic way. The term also refers toreducing the severity of a disease or symptoms associated with suchdisease prior to affliction with the disease. Such prevention orreduction of the severity of a disease prior to affliction refers toadministration of an antibody or pharmaceutical composition of thepresent invention to a subject that is not at the time of administrationafflicted with the disease. “Preventing” also refers to preventing therecurrence of a disease or of one or more symptoms associated with suchdisease. “Treatment” and “therapeutically,” refer to the act oftreating, as “treating” is defined above.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. In case of conflict, the present document, includingdefinitions, will control. Preferred methods and materials are describedbelow, although methods and materials similar or equivalent to thosedescribed herein can be used in practice or testing of the presentinvention. All publications, patent applications, patents and otherreferences mentioned herein are incorporated by reference in theirentirety to disclose and describe the methods and/or materials inconnection with which the publications are cited. The materials,methods, and examples disclosed herein are illustrative only and notintended to be limiting.

1. OPTICAL IMAGING SYSTEM

The present invention provides a compact and relatively low costapparatus for conducting a digital assay, such as a digital immunoassay,to detect and/or quantify an analyte of interest in a sample. Theapparatus may be handheld or positioned on a support surface orconnected to and supported by adjacent processing equipment. Theapparatus described herein provides a number of benefits overconventional optical imaging systems known in the art. For example, theapparatus of the present disclosure is compact, provides a largerimaging area, and includes a simplified structure, etc. As noted in FIG.1, an embodiment of the present invention is compared to a conventionalsystem (e.g., an optical microscope system). The size of the apparatusof the present invention is significantly smaller than a conventionalsystem. For example, one construction of the apparatus measures 12 cm×10cm×10 cm while the conventional system measures 70 cm×50 cm×50 cm. Inthis construction, the apparatus is about 5 times smaller than theconventional system. Additionally, the imaging area of the apparatus ofthe present invention is 100,000 sample chambers, which measures about30 mm², compared to 30,000 sample chambers, which measures about 9 mm²,for a conventional system. Image acquisition can occur under ambientconditions for embodiments of the present invention while theconventional system requires a dark room. Additional comparisons betweenembodiments of the present invention and a conventional system are shownin FIG. 1.

The construction of the apparatus of the present invention also is lesscomplicated than the construction of the conventional system. Theconventional system, which utilizes fluorescence imaging techniques fordetection of one or more analytes of interest in a sample, requiresmultiple optical filters for each of the two channels along withactuators for exchanging the filters. The present apparatus utilizes alight scattering imaging technique for analyte detection and a lightsource oriented at an angle relative to the sample array (comprising oneor more detection or reaction vessels) thus allowing use of a singlefilter and elimination of the multiple filters and the actuators thatare used in the conventional system. Additionally, the color of thelight source can be selected based on the emission filter that is usedfor analyte detection, thereby allowing use of a single filter foranalyte detection.

According to an embodiment, the apparatus comprises a number ofcomponents, including one or more detection or reaction vessels, a lightsource configured to emit light toward the detection vessel, a singlefilter positioned to receive light reflected from a sample in the one ormore detection vessels, that originated from the light source, andreceive an output from a sample in the detection vessel, and a detectorconfigured to receive a portion of the reflected light and a portion ofthe output that passes through the single filter.

FIGS. 2-5 illustrate an optical imaging system 100 according to anembodiment of the present invention. With reference to FIG. 4, theoptical imaging system 100 is configured to detect solid supports (suchas beads) that may contain an analyte of interest in a detection vessel104 (such as a sample array comprising a plurality of microwells ornanowells) and to detect an output from the sample in the detectionvessel 104 upon activation of the sample (such as by using a detectablelabel).

The optical imaging system 100 may include a support 108 for receivingthe detection vessel 104. The system 100 also includes a first lightsource 112 and may include a second light source 116. The first lightsource 112 may be comprised of a single light source or a plurality oflight sources. The first light source 112 is positioned above or belowthe detection vessel 104 and is oriented at an angle θ relative to anaxis 120 extending generally perpendicular to the detection vessel 104.For example, generally perpendicular includes 90 degrees+/−about 2degrees (i.e., 88 degrees to 92 degrees) relative to the detectionvessel 104. The angle θ is between about 0 degrees and about 90 degrees.In other embodiments, the angle θ is between about 45 degrees and about90 degrees. In another embodiment, the angle θ is 80 degrees.

The first light source 112 emits scattered light 124 toward thedetection vessel 104. The first light source 112 can comprise alight-emitting diode (LED) that emits light at a particular wavelength.For example, the first light source 112 can comprise a green LED thatemits light at about 520 nm. The scattered light 124 reflects off thesample in the detection vessel 104 as reflected light 128, which isreceived by a filter 132. The filter 132 allows a portion of thereflected light 128 through to a detector or camera 136. The camera 136generates an image, which presents bright pixels that visualize ordetermine positions of any solid supports (e.g., beads) containing theanalyte of interest in the image. The image(s) aids in the determinationof the number of detection vessels which contain a solid supportcontaining an analyte of interest and/or provide spatial informationregarding the position of the locations. See, for example, FIG. 6, whichillustrates images (A) and (D) generated with a first light source 112as described above. In other constructions, the color of the LED of thefirst light source 112 can be selected based on the filter 132 used inthe apparatus.

With continued reference to FIGS. 4 and 5, the second light source 116is positioned above or below the detection vessel 104 and is orientedgenerally perpendicular relative to the detection vessel 104. Forexample, generally perpendicular includes 90 degrees and + or − about 2degrees (i.e., 88 degrees to 92 degrees relative to the detection vessel104). In some embodiments, the second light source 116 is positionedalong the axis 120, while in other embodiments, the second light source116 is positioned adjacent to the axis 120. For example, as illustratedin FIG. 4 (Type A), the second light source 116 is laterally offset fromthe axis 120 so as to not disturb the scattered light reflected from thesamples and fluorescence emitted from samples (discussed below).

The second light source 116 emits excitation light 140 toward thedetection vessel 104. The second light source 116 can comprise a LEDthat emits excitation light at a particular wavelength. For example, thesecond light source 116 can comprise a blue LED that emits light atabout 450 nm. The excitation light 140 excites or activates the samplesin the detection vessel 104. For example, if a particular analyte ispresent in the sample(s), then the sample(s) emits an output 144, suchas a fluorescence. If the particular analyte is not present in thesample(s) then an output 144 is not produced. The filter 132 (same asthe filter that receives the reflected light 128) receives the output144 and allows the output 144 through to the camera 136. The camera 136generates an image, which presents bright pixels that visualize ordetermine which detection vessels contain the particular analyte ofinterest. See, for example, FIG. 6, which illustrates images (B) and (E)generated with a second light source 116 as described above. The secondlight source 116 can be specifically selected (e.g., LED color) based onthe filter 132 that is employed for analyte detection in the samples.

The camera 136 can be a CCD camera used to capture images. Otherexamples of cameras include charge injection devices (CIDs),complementary metal oxide semiconductor (CMOS) devices, scientific CMOS(sCMOS) devices, and time delay integration (TDI) devices.

The camera 136 can be electronically or communicatively coupled to acomputer 148. The computer 148 includes an electronic processor (forexample, a microprocessor, application-specific integrated circuit(ASIC), or another suitable electronic device), a storage device (forexample, a non-transitory, computer-readable storage medium), and acommunication interface, such as a transceiver, for communicating over acommunication network (e.g., wireless) and, optionally, one or moreadditional communication networks or connections. The electronicprocessor, the storage device, and the communication interfacecommunicate over one or more communication lines or buses. It should beunderstood that the computer may include additional components thanthose described above in various configurations and may performadditional functionality than the functionality described in the presentapplication. For example, in some embodiments, the functionalitydescribed herein as being performed by the computer may be distributedamong multiple devices, such as multiple computers and servers.

The electronic processor executes instructions stored in the storagedevice. In particular, the storage device stores an image analyzer. Theimage analyzer is a software application executable by the electronicprocessor. As described below, the image analyzer, when executed by theelectronic processor, communicates with the camera 136 over thecommunication networks (through the communication interface) to managemigration of data stored locally on the camera 136 to the one or moreremote storage locations (e.g., the storage device in the computer).

The image analyzer receives an input of images generated by the camera136. The image analyzer can process the images to combine them into amerged image illustrating bead and enzyme location of the samples in thedetection vessel 104. For example, see FIG. 6, which illustrates images(C) and (F) with analyzed and merged images. Specifically, image (C) isthe analyzed and merged image of images (A) and (B) in FIG. 6.Similarly, image (F) is the analyzed and merged image of images (D) and(E) in FIG. 6.

2. METHODS FOR ANALYTE ANALYSIS

Also provided herein are methods for analyte analysis. The method mayinvolve single molecule counting. In certain embodiments, a method foranalyte analysis may involve assessing an analyte of interest present ina sample. In certain embodiments, the assessing may be used fordetermining presence of and/or concentration of an analyte of interestin a sample. In certain embodiments, the method may also be used fordetermining presence of and/or concentration of a plurality of differentanalytes of interest present in a sample.

Provided herein are methods for detecting an analyte of interest inliquid droplet (wherein the analyte of interest is from a test orbiological sample). The method includes providing a first liquid dropletcontaining an analyte of interest, providing a second liquid dropletcontaining at least one solid support (such as, for example, a magneticsolid support (such as a bead)) which contains a specific binding memberthat binds to the analyte of interest, using energy to exert a force tomanipulate the first liquid droplet (which contains the analyte ofinterest) with the second liquid (containing the at least one solidsupport) to create a mixture (also referred to herein as a “reactionmixture”), moving all or at least a portion of the mixture to an arrayof wells (where one or more wells of the array are of sufficient size toaccommodate the at least one solid support), adding at least onedetectable label to the mixture before, after or both before or aftermoving a portion of the mixture to the array of wells and detecting theanalyte of interest in the wells. The array of wells is also referred toherein as a “detection vessel”.

In some embodiments, the wells can be a microwell array or nanowellarray. In some embodiments, the microwell array or nanowell array has adiameter of at least about 4 mm, at least about 5 mm, at least about 6mm, at least about 7 mm, at least about 8 mm, at least about 9 mm, or atleast about 10 mm. In some embodiments, the microwell array has adiameter of 6 mm. In some embodiments, the microwell array or nanowellarray contains approximately 100,000 to approximately 1,000,000 wells,approximately 200,000 to approximately 750,000 wells, or approximately300,000 to approximately 500,000 wells. In some embodiments, themicrowell array contains about 100,000, about 200,000, about 300,000,about 350,000, about 375,000, about 400,000, about 425,000, about450,000, about 475,000, about 500,000, about 600,000, about 700,000,about 800,000, about 900,000, or about 1,000,000 wells. In someembodiments, the microwell array contains 400,000 wells. In someembodiments, the wells can have at least about 1 μM diameter, at leastabout 2 μM diameter, at least about 3 μM diameter, at least about 4 μMdiameter, at least about 5 μM diameter, at least about 6 μM diameter, atleast about 7 μM diameter, at least about 8 μM diameter, at least about9 μM diameter, or at least about 10 μM diameter at the bottom of thewell. In some embodiments, the plurality of reaction vessels can be amicrowell array or nanowell array having a diameter of 6 mm andcontaining approximately 400,000 wells having a 5 μm diameter at thebottom of the well.

In certain embodiments, “using energy to exert a force to manipulate thefirst liquid droplet with the second liquid droplet” refers to the useof non-mechanical forces (namely, for example, energy created withoutthe use of pumps and/or valves) to provide or exert a force thatmanipulates (such as merges or combines) at least the first and secondliquid droplets (and optionally, additional droplets) into a mixture.Example of non-mechanical forces that can be used in the methodsdescribed herein include electric actuation force (such as dropletactuation, electrophoresis, electrowetting, dielectrophoresis,electrostatic actuation, electric field mediated, electrode mediated,capillary force, chromatography, centrifugation or aspiration) and/oracoustic force (such as surface acoustic wave (or “SAW”). In certainembodiments, the the electric actuation force generated is analternating current. For example, the alternating current can have aroot mean squared (rms) voltage of 10 V, 15 V, 20 V, 25 V, 30 V, 35V ormore. For example, such alternating current can have a rms voltage of 10V or more, 15 V or more, 20 V or more, 25 V or more, 30 V or more or 35V or more. Alternatively, the alternating current can have a frequencyin a radio frequency range.

In certain embodiments, if magnetic solid supports are used, an electricactuation force and a magnetic field can be applied and applied fromopposition directions, relative to the at least a portion of themixture. In certain other embodiments, the mixture is mixed by movingit: back and forth, in a circular pattern or by splitting it into two ormore submixtures and then merging the submixtures. In certain otherembodiments, an electric actuation force can be generated using a seriesor plurality of electrodes (namely, at least two or more, at least threeor more, at least four or more, at least five or more, at least six ormore, at least seven or more, at least eight or more, at least nine ormore, at least ten or more, at least eleven or more, at least twelve ormore, at least thirteen or more, at least fourteen or more, at leastfifteen or more, etc.) to move the mixture to the array of wells inorder to seal the wells (which are loaded with at least one solidsupport).

In certain embodiments, the moving of all or at least a portion of themixture to an array of wells results in the loading (filling and/orplacement) of the at least one solid support into the array of wells. Incertain embodiments, a magnetic field is used to facilitate movement ofthe mixture and thus, at least one solid support, into one or more wellsof the array. In certain embodiments, after the at least one solidsupports are loaded into the wells, any solid supports that are notloaded into a well can be removed using routine techniques known in theart. For example, such removing can involve generating an electricactuation force (such as that described previously herein) with a seriesor plurality of electrodes to move a fluid droplet (such as apolarizable fluid droplet) to the array of wells to move at least aportion of the mixture to a distance (the length of which is notcritical) from the array of wells. In certain embodiments, an aqueouswashing liquid can be used to remove the solid supports not bound to anyanalyte of interest. In such embodiments, the removal involvesgenerating an electric actuation force with a series or plurality ofelectrodes to move an aqueous wash (or washing) droplet (a thirddroplet) across the array of wells. The amount and type of aqueousliquid used for said washing is not critical.

In certain embodiments, the mixture in the method is an aqueous liquid.In other embodiments, the mixture is an immiscible liquid. In otherembodiments, the liquid droplet is a hydrophobic liquid droplet. Inother embodiments, the liquid droplet is a hydrophilic liquid droplet.In certain embodiments, the array of wells used in the method has ahydrophobic surface. In other embodiments, the array of wells has ahydrophilic surface.

In certain embodiments, the first liquid droplet used in the method is apolarizable liquid. In certain embodiments, the second liquid dropletused in the method is a polarizable liquid. In certain embodiments, thefirst and second liquid droplets used in the method are polarizableliquids. In certain embodiments, the mixture is a polarizable liquid. Incertain embodiments one or more of the first droplet, second droplet andmixture is a polarizable liquid.

In certain embodiments, the at least one solid support comprises atleast one specific binding member that specifically binds to the analyteof interest. In certain embodiments, the detectable label is added tothe mixture before moving at least a portion of the mixture to the arrayof wells. In certain other embodiments, the detectable label is added tothe mixture after the moving of at least a portion of the analyte ofinterest. In certain embodiments, the detectable label comprises atleast one specific binding member that specifically binds to the analyteof interest. In certain embodiments, the detectable label comprises achromagen, a florescent compound, an enzyme, a chemiluminescent compoundor a radioactive compound. In certain embodiments, the specific bindingmember is a receptor, aptamer or antibody. In certain embodiments, themethod further comprises positioning the at least a portion of themixture over the array of wells using a capillary element configured tofacilitate movement of the mixture to the array of wells.

In certain embodiments, when the mixture is a liquid and after themixture is moved to an array of wells, an aqueous phase is createdwithin the wells. In certain embodiments, the wells can be sealed by theaddition of one or more solvents (“solvent well sealing”). A hydrophilicor a hydrophobic solvent can be used. Hydrophilic solvents that can beused include hydrophilic alcohols, hydrophilic ethers, ketones, nitrilesolvents, dimethyl sulfoxides, and N,N-dimethylformamides, or mixturesthereof. Examples of hydrophilic alcohols include ethanol, methanol,propanol, and glycerin. Examples of hydrophilic ethers includetetrahydrofuran, polyethylene oxide, and 1,4-dioxane. Examples of ketoneincludes acetone and methyl ethyl ketone. Examples of the nitrilesolvents include acetonitrile. Hydrophobic solvents that can be usedinclude hydrocarbons, unsaturated hydrocarbons, aromatic hydrocarbons,silicone oils, perfluorocarbons, halogen solvents, hydrophobic ionicliquids and mixtures thereof. Examples of saturated hydrocarbons includealkanes, such as decane and hexadecane. Examples of unsaturatedhydrocarbon include squalene. Examples of aromatic hydrocarbon includebenzene and toluene. Examples of perfluorocarbon encompass Fluorinert®,FC-40, FC-72, FC-84, FC-77, FC-3255, FC-3283, FC-43, FC-70), 3M Novec4200, 3M Novec 4300, 3M FC-4432, 3M FC-4430, or 3M FC-4434. Examples ofhalogen solvents encompass chloroform, methylene chloride, andchlorobenzene. The hydrophobic ionic liquid denotes ionic liquid whichis not dissociated at least in water. Examples of ionic liquids include1-butyl-3-methylimidazolium hexafluorophosphate.

Because the hydrophilic or hydrophobic solvent has a density that isheavier than the aqueous phase, after the solvent is added, it movestowards the bottom of the well and displaces the aqueous phase, thusforcing it to the surface and creating a clear separation between theupper aqueous phase and the lower solvent phase. The upper aqueous phasecan be removed using routine techniques known in the art.

In certain embodiments, the method described herein is performed usingthe optical imaging system described in Section 1. In certainembodiments, the method described herein is performed using amicrofluidics device. In certain embodiments, the method describedherein is performed using a digital microfluidics device (DMF). Incertain embodiments, method described herein is performed using asurface acoustic wave based microfluidics device (SAW). In certainembodiments, method described herein is performed using an integratedDMF and analyte detection device. In certain embodiments, methoddescribed herein is performed using an integrated surface acoustic wavebased microfluidic device and analyte detection device. In certainembodiments, the methods described herein are performed using a roboticsbased assay processing unit.

Provided herein are methods for detecting an analyte of interest inliquid droplet (wherein the analyte of interest is from a test orbiological sample). The method includes providing a first liquid dropletcontaining an analyte of interest, providing a second liquid dropletcontaining at least one detectable label which contains a specificbinding member that binds to the analyte of interest, using energy toexert a force to manipulate the first liquid droplet (which contains theanalyte of interest) with the second liquid (containing the at least onesolid support) to create a mixture (namely, a reaction mixturecontaining an analyte/detectable label-specific binding member complex),moving all or at least a portion of the mixture to an array of wells(where one or more wells of the array are of sufficient size toaccommodate the at least one solid support), optionally sealing thewells using one or more solvents, and detecting the analyte of interestin the wells. In certain embodiments, “using energy to exert a force tomanipulate the first liquid droplet with the second liquid droplet”refers to the use of non-mechanical forces (namely, for example, energycreated without the use of pumps and/or valves) to provide or exert aforce that manipulates (such as merges or combines) at least the firstand second liquid droplets (and optionally, additional droplets) into amixture. Example of non-mechanical forces that can be used in themethods described herein include electric actuation force (such asdroplet actuation, electrophoresis, electrowetting, dielectrophoresis,electrostatic actuation, electric field mediated, electrode mediated,capillary force, chromatography, centrifugation or aspiration) and/oracoustic force (such as surface acoustic wave (or “SAW”). In certainembodiments, the electric actuation force generated is an alternatingcurrent. For example, the alternating current can have a root meansquared (rms) voltage of 10 V, 15 V, 20 V, 25 V, 30 V, 35V or more. Forexample, such alternating current can have a rms voltage of 10 V ormore, 15 V or more, 20 V or more, 25 V or more, 30 V or more or 35 V ormore. Alternatively, the alternating current can have a frequency in aradio frequency range.

In certain embodiments, the mixture is mixed by moving it back andforth, in a circular pattern or by splitting it into two or moresubmixtures and then merging the submixtures. In certain otherembodiments, an electric actuation force can be generated using a seriesor plurality of electrodes (namely, at least two or more, at least threeor more, at least four or more, at least five or more, at least six ormore, at least seven or more, at least eight or more, at least nine ormore, at least ten or more, at least eleven or more, at least twelve ormore, at least thirteen or more, at least fourteen or more, at leastfifteen or more, etc.) to move the mixture to the array of wells inorder to seal the wells (which are loaded with at least one solidsupport).

In certain embodiments, the moving of all or at least a portion of themixture to an array of wells results in the loading (filling and/orplacement) of the analyte/detectable label-specific binding membercomplex into the array of wells. In certain embodiments, a magneticfield is used to facilitate movement of the mixture and thus, at leastone analyte/detectable label-specific binding member complex into one ormore wells of the array. For example, such removing can involvegenerating an electric actuation force (such as that describedpreviously herein) with a series or plurality of electrodes to move afluid droplet (such as a polarizable fluid droplet) to the array ofwells to move at least a portion of the mixture to a distance (thelength of which is not critical) from the array of wells. In certainembodiments, an aqueous washing liquid can be used to remove anydetectable label-specific binding members not bound to any analyte. Insuch embodiments, the removal involves generating an electric actuationforce with a series or plurality of electrodes to move an aqueous wash(or washing) droplet (a third droplet) across the array of wells. Theamount and type of aqueous liquid used for said washing is not critical.

In certain embodiments, the mixture in the method is an aqueous liquid.In other embodiments, the mixture is an immiscible liquid. In otherembodiments, the liquid droplet is a hydrophobic liquid droplet. Inother embodiments, the liquid droplet is a hydrophilic liquid droplet.In certain embodiments, the array of wells used in the method has ahydrophobic surface. In other embodiments, the array of wells has ahydrophilic surface.

In certain embodiments, the first liquid droplet used in the method is apolarizable liquid. In certain embodiments, the second liquid dropletused in the method is a polarizable liquid. In certain embodiments, thefirst and second liquid droplets used in the method are polarizableliquids. In certain embodiments, the mixture is a polarizable liquid. Incertain embodiments one or more of the first droplet, second droplet andmixture is a polarizable liquid.

In certain embodiments, the detectable label is bound to at least onesolid support. In certain embodiments, the detectable label comprises achromagen, a florescent compound, an enzyme, a chemiluminescent compoundor a radioactive compound. In certain embodiments, the specific bindingmember is a receptor, aptamer or antibody. In certain embodiments, themethod further comprises positioning the at least a portion of themixture over the array of wells using a capillary element configured tofacilitate movement of the mixture to the array of wells.

In certain embodiments, when the mixture is a liquid and after themixture is moved to an array of wells, an aqueous phase is createdwithin the wells. In certain embodiments, the wells can be sealed by theaddition of one or more solvents as discussed previously herein.

In certain embodiments, the method described herein is performed usingthe compact digital immunoassay apparatus described herein. In certainembodiments, the method described herein is performed using amicrofluidics device. In certain embodiments, the method describedherein is performed using a digital microfluidics device (DMF). Incertain embodiments, method described herein is performed using asurface acoustic wave based microfluidics device (SAW). In certainembodiments, method described herein is performed using an integratedDMF and analyte detection device. In certain embodiments, methoddescribed herein is performed using an integrated surface acoustic wavebased microfluidic device and analyte detection device. In certainembodiments, method described herein is performed using a Robotics basedassay processing unit.

Provided herein are methods for measuring an analyte of interest inliquid droplet (wherein the analyte of interest is from a test orbiological sample). The method includes providing a first liquid dropletcontaining an analyte of interest, providing a second liquid dropletcontaining at least one solid support (such as, for example, a magneticsolid support (such as a bead)) which contains a specific binding memberthat binds to the analyte of interest, using energy to exert a force tomanipulate the first liquid droplet (which contains the analyte ofinterest) with the second liquid (containing the at least one solidsupport) to create a mixture (also referred to herein as a “reactionmixture”), moving all or at least a portion of the mixture to an arrayof wells (where one or more wells of the array are of sufficient size toaccommodate the at least one solid support), adding at least onedetectable label to the mixture before, after or both before or aftermoving a portion of the mixture to the array of wells, optionallysealing the array of wells using one or more solvents, and measuring theanalyte of interest in the wells. In certain embodiments, “using energyto exert a force to manipulate the first liquid droplet with the secondliquid droplet” refers to the use of non-mechanical forces (namely, forexample, energy created without the use of pumps and/or valves) toprovide or exert a force that manipulates (such as merges or combines)at least the first and second liquid droplets (and optionally,additional droplets) into a mixture. Example of non-mechanical forcesthat can be used in the methods described herein include electricactuation force (such as droplet actuation, electrophoresis,electrowetting, dielectrophoresis, electrostatic actuation, electricfield mediated, electrode mediated, capillary force, chromatography,centrifugation or aspiration) and/or acoustic force (such as surfaceacoustic wave (or “SAW”). In certain embodiments, the electric actuationforce generated is an alternating current. For example, the alternatingcurrent can have a root mean squared (rms) voltage of 10 V, 15 V, 20 V,25 V, 30 V, 35V or more. For example, such alternating current can havea rms voltage of 10 V or more, 15 V or more, 20 V or more, 25 V or more,30 V or more or 35 V or more. Alternatively, the alternating current canhave a frequency in a radio frequency range.

In certain embodiments, if magnetic solid supports are used, an electricactuation force and a magnetic field can be applied and applied fromopposition directions, relative to the at least a portion of themixture. In certain other embodiments, the mixture is mixed by movingit: back and forth, in a circular pattern or by splitting it into two ormore submixtures and then merging the submixtures. In certain otherembodiments, an electric actuation force can be generated using a seriesor plurality of electrodes (namely, at least two or more, at least threeor more, at least four or more, at least five or more, at least six ormore, at least seven or more, at least eight or more, at least nine ormore, at least ten or more, at least eleven or more, at least twelve ormore, at least thirteen or more, at least fourteen or more, at leastfifteen or more, etc.) to move the mixture to the array of wells inorder to seal the wells (which are loaded with at least one solidsupport).

In certain embodiments, the moving of all or at least a portion of themixture to an array of wells results in the loading (filling and/orplacement) of the at least one solid support into the array of wells. Incertain embodiments, a magnetic field is used to facilitate movement ofthe mixture and thus, at least one solid support, into one or more wellsof the array. In certain embodiments, after the at least one solidsupports are loaded into the wells, any solid supports that are notloaded into a well can be removed using routine techniques known in theart. For example, such removing can involve generating an electricactuation force (such as that described previously herein) with a seriesor plurality of electrodes to move a fluid droplet (such as apolarizable fluid droplet) to the array of wells to move at least aportion of the mixture to a distance (the length of which is notcritical) from the array of wells. In certain embodiments, an aqueouswashing liquid can be used to remove the solid supports not bound to anyanalyte of interest. In such embodiments, the removal involvesgenerating an electric actuation force with a series or plurality ofelectrodes to move an aqueous wash (or washing) droplet (a thirddroplet) across the array of wells. The amount and type of aqueousliquid used for said washing is not critical.

In certain embodiments, the mixture in the method is an aqueous liquid.In other embodiments, the mixture is an immiscible liquid. In otherembodiments, the liquid droplet is a hydrophobic liquid droplet. Inother embodiments, the liquid droplet is a hydrophilic liquid droplet.In certain embodiments, the array of wells used in the method have ahydrophobic surface. In other embodiments, the array of wells has ahydrophilic surface.

In certain embodiments, the first liquid droplet used in the method is apolarizable liquid. In certain embodiments, the second liquid dropletused in the method is a polarizable liquid. In certain embodiments, thefirst and second liquid droplets used in the method are polarizableliquids. In certain embodiments, the mixture is a polarizable liquid. Incertain embodiments one or more of the first droplet, second droplet andmixture is a polarizable liquid.

In certain embodiments, the at least one solid support comprises atleast one specific binding member that specifically binds to the analyteof interest. In certain embodiments, the detectable label is added tothe mixture before moving at least a portion of the mixture to the arrayof wells. In certain other embodiments, the detectable label is added tothe mixture after the moving of at least a portion of the analyte ofinterest to the array of wells. In certain embodiments, the detectablelabel comprises at least one specific binding member that specificallybinds to the analyte of interest. In certain embodiments, the detectablelabel comprises a chromagen, a florescent compound, an enzyme, achemiluminescent compound or a radioactive compound. In certainembodiments, the specific binding member is a receptor, aptamer orantibody. In certain embodiments, the method further comprisespositioning the at least a portion of the mixture over the array ofwells using a capillary element configured to facilitate movement of themixture to the array of wells.

In certain embodiments, when the mixture is a liquid and after themixture is moved to an array of wells, an aqueous phase is createdwithin the wells. In certain embodiments, the wells can be sealed by theaddition of one or more solvents as discussed previously herein.

In certain embodiments, the method described herein is performed usingthe optical imaging system described in Section 1. In certainembodiments, the method described herein is performed using amicrofluidics device. In certain embodiments, the method describedherein is performed using a digital microfluidics device (DMF). Incertain embodiments, method described herein is performed using asurface acoustic wave based microfluidics device (SAW). In certainembodiments, method described herein is performed using an integratedDMF and analyte detection device. In certain embodiments, methoddescribed herein is performed using an integrated surface acoustic wavebased microfluidic device and analyte detection device. In certainembodiments, method described herein is performed using a Robotics basedassay processing unit.

In certain embodiments, the measuring first involves determining thetotal number of solid supports in the well of the array (“total solidsupport number”). Next, the number of solid supports in the wells of thearray that contain the detectable label are determined, such as, forexample, determining the intensity of the signal produced by thedetectable label (“positives”). The positives are subtracted from thetotal solid support number to provide the number of solid supports inthe array of wells that do not contain a detectable label or are notdetected (“negatives”). Then, the ratio of positives to negatives in thearray of wells can be determined and then compared to a calibrationcurve. Alternatively, digital quantitation using the Poisson equationP(x; μ) as shown below:

P(x; μ)=(e ^(−μ))(μ^(x))/x!

where:

e: A is a constant equal to approximately 2.71828,

μ: is the mean number of successes that occur in a specified region, and

x: is the actual number of successes that occur in a specified region.

The sample used in the methods described herein may be any test samplecontaining or suspected of containing an analyte of interest. As usedherein, “analyte”, “target analyte”, “analyte of interest” are usedinterchangeably and refer to the analyte being measured in the methodsand devices disclosed herein. Analytes of interest are further describedbelow.

“Contacting” and grammatical equivalents thereof as used herein refer toany type of combining action which brings a specific binding member intosufficiently close proximity with the analyte of interest in the samplesuch that a binding interaction will occur if the analyte of interestspecific for the specific binding member is present in the sample.Contacting may be achieved in a variety of different ways, includingcombining the sample with a specific binding member, exposing a targetanalyte to a specific binding member by introducing the binding memberin close proximity to the analyte, and the like.

In certain cases, the first specific binding member may be immobilizedon a solid support. As used herein, the term “immobilized” refers to astable association of the first specific binding member with a surfaceof a solid support. By “stable association” is meant a physicalassociation between two entities in which the mean half-life ofassociation is one day or more, e.g., under physiological conditions. Incertain aspects, the physical association between the two entities has amean half-life of two days or more, one week or more, one month or more,including six months or more, e.g., 1 year or more, in PBS at 4° C.According to certain embodiments, the stable association arises from acovalent bond between the two entities, a non-covalent bond between thetwo entities (e.g., an ionic or metallic bond), or other forms ofchemical attraction, such as hydrogen bonding, Van der Waals forces, andthe like.

The solid support having a surface on which the specific binding memberis immobilized may be any convenient surface in planar or non-planarconformation, such as a surface of a microfluidic chip, an interiorsurface of a chamber, an exterior surface of a bead (as defined herein),or an interior and/or exterior surface of a porous bead. For example,the first specific binding member may be attached covalently ornon-covalently to a bead, e.g., latex, agarose, sepharose, streptavidin,tosylactivated, epoxy, polystyrene, amino bead, amine bead, carboxylbead, or the like. In certain embodiments, the bead may be a particle,e.g., a microparticle. In some embodiments, the microparticle may bebetween about 0.1 nm and about 10 microns, between about 50 nm and about5 microns, between about 100 nm and about 1 micron, between about 0.1 nmand about 700 nm, between about 500 nm and about 10 microns, betweenabout 500 nm and about 5 microns, between about 500 nm and about 3microns, between about 100 nm and 700 nm, or between about 500 nm and700 nm. For example, the microparticle may be about 4-6 microns, about2-3 microns, or about 0.5-1.5 microns. Particles less than about 500 nmare sometimes considered nanoparticles. Thus, the microparticleoptionally may be a nanoparticle between about 0.1 nm and about 500 nm,between about 10 nm and about 500 nm, between about 50 nm and about 500nm, between about 100 nm and about 500 nm, about 100 nm, about 150 nm,about 200 nm, about 250 nm, about 300 nm, about 350 nm, about 400 nm,about 450 nm, or about 500 nm.

In certain embodiments, the bead may be a magnetic bead or a magneticparticle. In certain embodiments, the bead may be a magnetic nanobead,nanoparticle, microbead or microparticle. Magnetic beads/particles maybe ferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic orferrofluidic. Exemplary ferromagnetic materials include Fe, Co, Ni, Gd,Dy, CrO₂, MnAs, MnBi, EuO, NiO/Fe. Examples of ferrimagnetic materialsinclude NiFe₂O₄, CoFe₂O₄, Fe₃O₄ (or FeO.Fe₂O₃). Beads can have a solidcore portion that is magnetic and is surrounded by one or morenon-magnetic layers. Alternately, the magnetic portion can be a layeraround a non-magnetic core. The solid support on which the firstspecific binding member is immobilized may be stored in dry form or in aliquid. The magnetic beads may be subjected to a magnetic field prior toor after contacting with the sample with a magnetic bead on which thefirst specific binding member is immobilized.

After the contacting step, the sample and the first specific bindingmember may be incubated for a sufficient period of time to allow for thebinding interaction between the specific binding member and analyte tooccur. In addition, the incubating may be in a binding buffer thatfacilitates the specific binding interaction. The binding affinityand/or specificity of the first specific binding member and/or thesecond specific binding member may be manipulated or altered in theassay by varying the binding buffer. In some embodiments, the bindingaffinity and/or specificity may be increased by varying the bindingbuffer. In some embodiments, the binding affinity and/or specificity maybe decreased by varying the binding buffer.

The binding affinity and/or specificity of the first specific bindingmember and/or the second specific binding member may be measured usingthe disclosed methods and device described below. In some embodiments,the one aliquot of sample is assayed using one set of conditions andcompared to another aliquot of sample assayed using a different set ofconditions, thereby determining the effect of the conditions on thebinding affinity and/or specificity. For instance, changing or alteringthe condition can be one or more of removing the target analyte from thesample, adding a molecule that competes with the target analyte or theligand for binding, and changing the pH, salt concentration, ortemperature. Additionally or alternatively, a duration of time can bethe variable and changing the condition may include waiting for aduration of time before again performing the detection methods.

The binding buffer may include molecules standard for antigen-antibodybinding buffers such as, albumin (e.g., BSA), non-ionic detergents(Tween-20, Triton X-100), and/or protease inhibitors (e.g., PMSF). Incertain cases, the binding buffer may be added to the microfluidic chip,chamber, etc., prior to or after adding the sample. In certain cases,the first specific binding member may be present in a binding bufferprior to contacting with the sample. The length of time for bindinginteraction between the binding member and analyte to occur may bedetermined empirically and may depend on the binding affinity andbinding avidity between the binding member and the analyte. In certainembodiments, the contacting or incubating may be for a period of 5 secto 1 hour, such as, 10 sec-30 minutes, or 1 minute-15 minutes, or 5minutes-10 minutes, e.g., 10 sec, 15 sec, 30 sec, 1 minute, 5 minutes,10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour or 2 hours. Otherconditions for the binding interaction, such as, temperature, saltconcentration, may also be determined empirically or may be based onmanufacturer's instructions. For example, the contacting may be carriedout at room temperature (21° C.-28° C., e.g., 23° C.-25° C.), 37° C., or4° C. In certain embodiments, an optional mixing of the sample with thefirst specific binding member may be carried out during the contactingstep.

Following complex formation between the immobilized first specificbinding member and the analyte, any unbound analyte may be removed fromthe vicinity of the first specific binding member along with the samplewhile the complex of the first specific binding member and the analytemay be retained due to its association with the solid support.Optionally, the solid support may be contacted with a wash buffer toremove any molecules non-specifically bound to the solid support.

After the first contacting step, and the optional removal of sampleand/or optional wash steps, the complex of the first specific bindingmember and the analyte may be contacted with a second specific bindingmember, thereby leading to the formation of a sandwich complex in whichthe analyte is bound by the two specific binding members. An optionalmixing of the second member with the first specific bindingmember-analyte complex may be carried out during the second contactingstep. In some embodiments, immobilization of the analyte molecules withrespect to a surface may aid in removal of any excess second specificbinding members from the solution without concern of dislodging theanalyte molecule from the surface. In some embodiments, the secondspecific binding member may include a detectable label comprising one ormore signal-producing substances, such as chromagens, fluorescentcompounds, chemiluminescent compounds, enzymes, radioactive compounds,and the like.

As noted above, the second contacting step may be carried out inconditions sufficient for binding interaction between the analyte andthe second specific binding member. Following the second contactingstep, any unbound second specific binding member may be removed,followed by an optional wash step. Any unbound second specific bindingmember may be separated from the complex of the first specific bindingmember-analyte-second specific binding member by a suitable means suchas, droplet actuation, electrophoresis, electrowetting,dielectrophoresis, electrostatic actuation, electric field mediated,electrode mediated, capillary force, chromatography, centrifugation,aspiration or SAW. Upon removal of any unbound second specific bindingmember from the vicinity of the complex of the first specific bindingmember-analyte-second specific binding member, the detectable labelattached to the second specific binding member present in the complex ofthe first specific binding member-analyte-second specific binding membermay be separated by a suitable means or may be detected using techniquesknown in the art. In some embodiments, the detectable label comprises adetectable label comprising one or more signal-producing substances,such as chromagens, fluorescent compounds, enzymes, chemiluminescentcompounds, radioactive compounds, and the like. Alternatively, in someembodiments, if the detectable label comprises a tag, the tag can becleaved or disassociated from the complex which remains after removal ofunbound reagents. For example, the tag may be attached to the secondspecific binding member via a cleavable linker (“cleavable linker” asdescribed herein. The complex of the first specific bindingmember-analyte-second specific binding member may be exposed to acleavage agent that mediates cleavage of the cleavable linker.

As noted herein, the tag may include a nucleic acid. In certainembodiments, the quantification of the analyte does not includedetermining the identity of the tag by determining identity of at leasta portion of the nucleic acid sequence present in the tag. For example,the counting step may not include determining a sequence of the tag. Inother embodiments, the tag may not be sequenced, however, identity ofthe tag may be determined to the extent that one tag may bedistinguished from another tag based on a differentiable signalassociated with the tag due its size, conformation, charge, amount ofcharge and the like. Identification of tag may be useful in methodsinvolving simultaneous analysis of a plurality of different analytes ina sample, for example, two, three, four, or more different analytes in asample.

In certain embodiments, the simultaneous analysis of multiple analytesin a single sample may be performed by using a plurality of differentfirst and second specific binding members where a pair of first andsecond specific binding members is specific to a single analyte in thesample. In these embodiments, the detectable label associated with thesecond specific binding member of a first pair of first and secondspecific binding members specific to a single analyte may bedistinguishable from the detectable label associated with the secondspecific binding member of a second pair of first and second specificbinding members specific to a different analyte. As noted above, a firstdetectable label may be distinguishable from second detectable labelbased on difference in signal-producing substances, etc.

In some embodiments, the concentration of an analyte in the fluid samplethat may be substantially accurately determined is less than about 5000fM (femtomolar), less than about 3000 fM, less than about 2000 fM, lessthan about 1000 fM, less than about 500 fM, less than about 300 fM, lessthan about 200 fM, less than about 100 fM, less than about 50 fM, lessthan about 25 fM, less than about 10 fM, less than about 5 fM, less thanabout 2 fM, less than about 1 fM, less than about 500 aM (attomolar),less than about 100 aM, less than about 10 aM, less than about 5 aM,less than about 1 aM, less than about 0.1 aM, less than about 500 zM(zeptomolar), less than about 100 zM, less than about 10 zM, less thanabout 5 zM, less than about 1 zM, less than about 0.1 zM, or less.

In some cases, the limit of detection (e.g., the lowest concentration ofan analyte which may be determined in solution) is about 100 fM, about50 fM, about 25 fM, about 10 fM, about 5 fM, about 2 fM, about 1 fM,about 500 aM (attomolar), about 100 aM, about 50 aM, about 10 aM, about5 aM, about 1 aM, about 0.1 aM, about 500 zM (zeptomolar), about 100 zM,about 50 zM, about 10 zM, about 5 zM, about 1 zM, about 0.1 zM, or less.In some embodiments, the concentration of analyte in the fluid samplethat may be substantially accurately determined is between about 5000 fMand about 0.1 fM, between about 3000 fM and about 0.1 fM, between about1000 fM and about 0.1 fM, between about 1000 fM and about 0.1 zM,between about 100 fM and about 1 zM, between about 100 aM and about 0.1zM, or less.

The upper limit of detection (e.g., the upper concentration of ananalyte which may be determined in solution) is at least about 100 fM,at least about 1000 fM, at least about 10 pM (picomolar), at least about100 pM, at least about 100 pM, at least about 10 nM (nanomolar), atleast about 100 nM, at least about 1000 nM, at least about 10 μM, atleast about 100 μM, at least about 1000 μM, at least about 10 mM, atleast about 100 mM, at least about 1000 mM, or greater.

In some cases, the presence and/or concentration of the analyte in asample may be detected rapidly, usually in less than about 1 hour, e.g.,45 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes, 1 minute, or30 seconds.

In certain embodiments, at least some of the methods described hereinmay be performed using the optical imaging system described inSection 1. In certain embodiments, at least some steps of the methodsdescribed herein may be carried out on a digital integratedmicrofluidics and analyte detection device, such as the device describedherein. In certain embodiments, the methods of the present disclosureare carried out using a digital integrated microfluidics device inconjunction with an analyte detection device. For example, the digitalmicrofluidics device and the analyte detection device may be separatedevices and a droplet containing the detectable label may be generatedin the microfluidics device and transported to the analyte detectiondevice.

In certain embodiments, the methods of the present disclosure arecarried out using a device in which a digital microfluidics module isintegrated with an analyte detection device, such as the devicedescribed below. In certain embodiments, the digital integratedmicrofluidics module and the analyte detection device may be reversiblyintegrated. For example, the two modules may be combined physically toform the integrated device and which device could then be separated intothe individual modules. In certain embodiments, the methods of thepresent disclosure are carried out using a disposable cartridge thatincludes a microfluidics module with a built-in analyte detectiondevice. Exemplary embodiments of the devices used for performing themethods provided herein are described further in the next section.

Exemplary embodiments of the present method include merging a sampledroplet containing an analyte of interest with a droplet containing afirst specific binding member that binds to the analyte of interest andthat may be immobilized on a solid support (such as magnetic particlesor beads). The single merged droplet can be incubated for a period oftime sufficient to allow binding of the first specific binding member tothe analyte of interest. Optionally, the single droplet may be agitatedto facilitate mixing of the sample with the first specific bindingmember. Mixing may be achieved by moving the single droplet back andforth, moving the single droplet around over a plurality of electrodes,splitting a droplet and then merging the droplets, or using SAWs, andthe like. Next, the single droplet may be subjected to a magnetic forceto retain the beads at a location in the device while the droplet may bemoved away and replaced with a droplet containing a second specificbinding member, which second specific binding member can optionallycontain a detectable label. An optional wash step may be performed,prior to adding the second specific binding member, by moving a dropletof wash buffer to the location at which the beads are retained using themagnetic force. After a period of time sufficient for the secondspecific binding member to bind the analyte bound to the first specificbinding member, the droplet containing the second specific bindingmember may be moved away while the beads are retained at the firstlocation. The beads may be washed using a droplet of wash buffer.Following the wash step, the magnetic force may be removed and thedroplet containing labeled beads (containing the first specific bindingmember/analyte/second specific binding member-an optional detectablelabel) are moved to a detection module such as that described herein.The labeled beads are allowed to settle into an array of wells in thedetection module. The beads may settle via gravitational force or byapplying electric or magnetic force. Following a wash step to remove anybeads not located inside the wells, the wells may be sealed using asolvent (such as a hydrophobic liquid, such as an oil). In the aboveembodiments, optionally, after the combining, a droplet may bemanipulated (e.g., moved back and forth, moved in a circular direction,oscillated, split/merged, exposed to SAW, etc.) to facilitate mixing ofthe sample with the assay reagents, such as, the first specific bindingmember, second specific binding member, etc. In embodiments where thedetectable label is an enzyme, a substrate can be added either before orafter moving the complex is moved to the array of wells.

The moving of the droplets in the integrated microfluidic and analytedetection device may be carried out using electrical force (e.g.,electrowetting, dielectrophoresis, electrode-mediated,opto-electrowetting, electric-field mediated, and electrostaticactuation) pressure, surface acoustic waves and the like. The force usedfor moving the droplets may be determined based on the specifics of thedevice, which are described in the following sections, and for theparticular device described herein.

In some embodiments, one or more detected signals corresponds to abinding event of a specific binding member to an analyte. In someembodiments, one detected signal corresponds to a binding event of aspecific binding member to an analyte. In some embodiments, two or moredetected signals correspond to a binding event of a specific bindingmember to an analyte.

In some embodiments, the solid support comprising the first specificbinding member and second specific binding member are added sequentiallyor simultaneously to the sample.

The detection of the analyte is correlated by the detectable product ordetectable label, namely, a signal, generated by the at least one signalgenerating compound and the at least one signal generating substrate. Insome embodiments, the at least one signal generating compound is anenzyme and the at least one signal generating substrate is a substratefor the enzyme. In some embodiments, the substrate for the enzyme is acolorimetric, fluorogenic (non-fluorescent) substrate or a chromogenicsubstrate. In some embodiments, the detectable signal is a fluorescentsignal. For example, the enzyme may be a, polynucleotidase, arginase,adenase, aminopolypeptidase, pepsin, lipases, catalase, tyrosinases,alcohol dehydrogenase, succinic dehydrogenase, diaphorase, glyoxalase,aldolase, glucose oxidase, horseradish peroxidase, galactosidase (suchas beta-galactosidase), phosphatases, phosphorylases and hexokinases orcombinations thereof. Examples of enzymatic substrates that can be usedinclude a chemiluminescent substrate such as CDP-Star®, (disodium4-chloro-3-(methoxyspiro{1,2-dioxetane-3,2′-(5′-chloro)tricyclo[3.3.1.1.s-up.3,7]decane}-4-yl)phenylphosphate), CSPD®, or (disodium3-(4-methoxyspiro{1,2-dioxetane-3,2-(5′-chloro)tricyclo[3.3.1.1-.sup.3,7]decane}-4-yl)phenylphosphate); a luminescent substrate such as p-nitrophenyl phosphate,5-bromo-4-chloro-3-indolyl phosphate (BCIP), 4-nitro blue tetrazoliumchloride (NBT), or iodonitrotetrazolium (INT); a fluorescent substratesuch as 4-methylumbelliferyl phosphate (4-MUP); and a chromogenicsubstrate such as 5-bromo-4-chloro-3-indolyl phosphate (BCIP), disodium5-bromo-6-chloro-indolyl phosphate, or p-nitrophenyl phosphate.

In some aspects, enzymes that can be used include those which contain aninhibitor molecule (such as a protein, peptide, etc.) bound to a siteother than the active binding site of the enzyme. Such inhibitormolecules change the conformation of the active binding site of theenzyme and prevent it from binding to the substrate. Examples ofinhibitor molecules include protease inhibitors. The inhibitor can beremoved from the enzyme using routine techniques known in the art toallow the enzyme to bind to the substrate thus allowing a signalgenerating reaction to occur.

In some embodiments, the enzyme can convert a non-fluorescent substrateinto a fluorescent substrate. In some embodiments, the enzyme cangenerate color using a chromogenic substrate.

3. SPECIFIC BINDING MEMBERS

As will be appreciated by those in the art, the specific binding memberswill be determined by the analyte to be analyzed. Specific bindingmembers for a wide variety of target molecules are known or can bereadily found or developed using known techniques. For example, when thetarget analyte is a protein, the specific binding members may includeproteins, particularly antibodies or fragments thereof (e.g.,antigen-binding fragments (Fabs), Fab′ fragments, F(ab′)₂ fragments,recombinant antibodies, chimeric antibodies, single-chain Fvs (“scFv”),single chain antibodies, single domain antibodies, such as variableheavy chain domains (“VHH”; also known as “VHH fragments”) derived fromanimals in the Camelidae family (VHH and methods of making them aredescribed in Gottlin et al., Journal of Biomolecular Screening, 14:77-85(2009)), recombinant VHH single-domain antibodies, and VNAR fragments,disulfide-linked Fvs (“sdFv”), and anti-idiotypic (“anti-Id”)antibodies, and functionally active epitope-binding fragments of any ofthe above, full-length polyclonal or monoclonal antibodies,antibody-like fragments, etc.), other proteins, such as receptorproteins, Protein A, Protein C, or the like. In case where the analyteis a small molecule, such as, steroids, bilins, retinoids, and lipids,the first and/or the second specific binding member may be a scaffoldprotein (e.g., lipocalins) or a receptor. In some cases, specificbinding member for protein analytes may be a peptide. For example, whenthe target analyte is an enzyme, suitable specific binding members mayinclude enzyme substrates and/or enzyme inhibitors which may be apeptide, a small molecule and the like. In some cases, when the targetanalyte is a phosphorylated species, the specific binding members maycomprise a phosphate-binding agent. For example, the phosphate-bindingagent may comprise metal-ion affinity media such as those describe inU.S. Pat. No. 7,070,921 and U.S. Patent Application No. 2006/0121544.

When the target molecule is a carbohydrate, potentially suitable capturecomponents (as defined herein) include, for example, antibodies,lectins, and selectins. As will be appreciated by those of ordinaryskill in the art, any molecule that can specifically associate with atarget molecule of interest may potentially be used as a specificbinding member.

For certain embodiments, suitable target analyte/specific binding membercomplexes can include, but are not limited to, antibodies/antigens,antigens/antibodies, receptors/ligands, ligands/receptors,proteins/nucleic acid, enzymes/substrates and/or inhibitors,carbohydrates (including glycoproteins and glycolipids)/lectins and/orselectins, proteins/proteins, proteins/small molecules, etc.

In a particular embodiment, the first specific binding member and/orsecond specific binding member may be attached to a solid support via alinkage, which may comprise any moiety, functionalization, ormodification of the support and/or specific binding member thatfacilitates the attachment of the specific binding member to thesupport. The linkage between the specific binding member and the supportmay include one or more chemical or physical (e.g., non-specificattachment via van der Waals forces, hydrogen bonding, electrostaticinteractions, hydrophobic/hydrophilic interactions; etc.) bonds and/orchemical spacers providing such bond(s).

In certain embodiments, a solid support may also comprise a protective,blocking, or passivating layer that can eliminate or minimizenon-specific attachment of non-capture components (e.g., analytemolecules, specific binding members) to the binding surface during theassay which may lead to false positive signals during detection or toloss of signal. Examples of materials that may be utilized in certainembodiments to form passivating layers include, but are not limited to:polymers, such as poly(ethylene glycol), that repel the non-specificbinding of proteins; naturally occurring proteins with this property,such as serum albumin and casein; surfactants, e.g., zwitterionicsurfactants, such as sulfobetaines; naturally occurring long-chainlipids; polymer brushes, and nucleic acids, such as salmon sperm DNA.

Certain embodiments utilize specific binding members that are proteinsor polypeptides. As is known in the art, any number of techniques may beused to attach a polypeptide to a wide variety of solid supports. A widevariety of techniques are known to add reactive moieties to proteins,for example, the method outlined in U.S. Pat. No. 5,620,850. Further,methods for attachment of proteins to surfaces are known, for example,see Heller, Acc. Chem. Res. 23:128 (1990).

As explained herein, binding between the specific binding members andthe analyte, is specific, e.g., as when the specific binding member andthe analyte are complementary parts of a binding pair. In certainembodiments, the specific binding member binds specifically to theanalyte. By “specifically bind” or “binding specificity,” it is meantthat the specific binding member binds the analyte molecule withspecificity sufficient to differentiate between the analyte molecule andother components or contaminants of the test sample. For example, thespecific binding member, according to one embodiment, may be an antibodythat binds specifically to an epitope on an analyte. The antibody,according to one embodiment, can be any antibody capable of bindingspecifically to an analyte of interest. For example, appropriateantibodies include, but are not limited to, monoclonal antibodies,bispecific antibodies, minibodies, domain antibodies (dAbs) (e.g., suchas described in Holt et al. (2014) Trends in Biotechnology 21:484-490),and including single domain antibodies sdAbs that are naturallyoccurring, e.g., as in cartilaginous fishes and camelid, or which aresynthetic, e.g., nanobodies, VHH, or other domain structure), syntheticantibodies (sometimes referred to as antibody mimetics), chimericantibodies, humanized antibodies, antibody fusions (sometimes referredto as “antibody conjugates”), and fragments of each, respectively. Asanother example, the analyte molecule may be an antibody and the firstspecific binding member may be an antigen and the second specificbinding member may be a secondary antibody that specifically binds tothe target antibody or the first specific binding member may be asecondary antibody that specifically binds to the target antibody andthe second specific binding member may be an antigen.

In some embodiments, the specific binding member may be chemicallyprogrammed antibodies (cpAbs) (described in Rader (2014) Trends inBiotechnology 32:186-197), bispecific cpAbs, antibody-recruitingmolecules (ARMs) (described in McEnaney et al. (2012) ACS Chem. Biol.7:1139-1151), branched capture agents, such as a triligand capture agent(described in Millward et al. (2011) J. Am. Chem. Soc. 133:18280-18288),engineered binding proteins derived from non-antibody scaffolds, such asmonobodies (derived from the tenth fibronectin type III domain of humanfibronectin), affibodies (derived from the immunoglobulin bindingprotein A), DARPins (based on Ankyrin repeat modules), anticalins(derived from the lipocalins bilin-binding protein and human lipocalin2), and cysteine knot peptides (knottins) (described in Gilbreth andKoide, (2012) Current Opinion in Structural Biology 22:1-8; Banta et al.(2013) Annu. Rev. Biomed. Eng. 15:93-113), WW domains (described inPatel et al. (2013) Protein Engineering, Design & Selection26(4):307-314), repurposed receptor ligands, affitins (described inBéhar et al. (2013) 26:267-275), and/or Adhirons (described in Tiede etal. (2014) Protein Engineering, Design & Selection 27:145-155).

According to one embodiment in which an analyte is a biological cell(e.g., mammalian, avian, reptilian, other vertebrate, insect, yeast,bacterial, cell, etc.), the specific binding members may be ligandshaving specific affinity for a cell surface antigen (e.g., a cellsurface receptor). In one embodiment, the specific binding member may bean adhesion molecule receptor or portion thereof, which has bindingspecificity for a cell adhesion molecule expressed on the surface of atarget cell type. In use, the adhesion molecule receptor binds with anadhesion molecule on the extracellular surface of the target cell,thereby immobilizing or capturing the cell, the bound cell may then bedetected by using a second specific binding member that may be the sameas the first specific binding member or may bind to a different moleculeexpressed on the surface of the cell.

In some embodiments, the binding affinity between analyte molecules andspecific binding members should be sufficient to remain bound under theconditions of the assay, including wash steps to remove molecules orparticles that are non-specifically bound. In some cases, for example inthe detection of certain biomolecules, the binding constant of theanalyte molecule to its complementary specific binding member may bebetween at least about 10⁴ and about 10⁶ M⁻¹, at least about 10⁵ andabout 10⁹ M⁻¹, at least about 10⁷ and about 10⁹ M⁻¹, greater than about10⁹ M⁻¹, or greater.

4. EXEMPLARY TARGET ANALYTES

As will be appreciated by those in the art, any analyte that can bespecifically bound by a first specific binding member and a secondspecific binding member may be detected and, optionally, quantifiedusing methods and devices of the present disclosure.

In some embodiments, the analyte may be a biomolecule or biologicalmolecule. Non-limiting examples of biomolecules and biological moleculesinclude macromolecules such as, proteins, lipids, and carbohydrates. Incertain instances, the analyte may be hormones, antibodies, growthfactors, cytokines, enzymes, receptors (e.g., neural, hormonal,nutrient, and cell surface receptors) or their ligands, cancer markers(e.g., PSA, TNF-alpha), markers of myocardial infarction (e.g.,troponin, creatine kinase, BNP, pro-BNP, NT-ProBNP, CK-MB, Galectin-3,and the like), thyroid markers (e.g., Anti-Tg, Anti-TPO, Free T3, FreeT4, T-uptake, Total T3, Total T4, TSH), toxins, drugs (e.g., drugs ofaddiction), metabolic agents (e.g., including vitamins), and the like.Non-limiting embodiments of protein analytes include peptides,polypeptides, protein fragments, protein complexes, fusion proteins,recombinant proteins, phosphoproteins, glycoproteins, lipoproteins, orthe like. In some embodiments, the analyte may be a biomarker, such as abiomarker for traumatic brain injury, sepsis, or coagulation, an analyteinvolved with general chemistry (e.g., ammonia, AST, cholesterol, etc.),a protein (e.g., transferrin, CRP, etc.), an analyte for therapeuticdrug monitoring (e.g., Methotrexate), an analyte for transplant (e.g.,tacrolimus), a drug of abuse, or a biomarker for genetic disorders.

In certain embodiments, the analyte may be a post-translationallymodified protein (e.g., phosphorylated, methylated, glycosylatedprotein) and the first or the second specific binding member may be anantibody specific to a post-translational modification. A modifiedprotein may be bound to a first specific binding member immobilized on asolid support where the first specific binding member binds to themodified protein but not the unmodified protein. In other embodiments,the first specific binding member may bind to both the unmodified andthe modified protein, and the second specific binding member may bespecific to the post-translationally modified protein.

In some embodiments, the analyte may be a cell, such as, circulatingtumor cell, pathogenic bacteria, viruses (including retroviruses,herpesviruses, adenoviruses, lentiviruses, Filoviruses (e.g., West Nile,Ebola and Zika viruses), hepatitis viruses (e.g., A, B, C, D, and E);HPV, Parvovirus, etc.; spores, etc.

A non-limiting list of analytes that may be analyzed by the methodspresented herein include Aβ42 amyloid beta-protein, fetuin-A, tau,secretogranin II, prion protein, Alpha-synuclein, tau protein,neurofilament light chain, parkin, PTEN induced putative kinase 1, DJ-1,leucine-rich repeat kinase 2, mutated ATP13A2, Apo H, ceruloplasmin,Peroxisome proliferator-activated receptor gamma coactivator-1 alpha(PGC-1α), transthyretin, Vitamin D-binding Protein, Active-B12, B12,cortisol, folate, frustosamine, homocysteine, intact PTH, pepsinogen I &II, DHEA-S, Estradiol, hCG, progesterone, prolactin, SHBG, testosterone,proapoptotic kinase R (PKR) and its phosphorylated PKR (pPKR), IL-12p40,CXCL13, IL-8, Dkk-3 (semen), p14 endocan fragment, Serum, ACE2,autoantibody to CD25, hTERT, CAI25 (MUC 16), VEGF, sIL-2, Osteopontin,Human epididymis protein 4 (HE4), Alpha-Fetoprotein, Albumin,albuminuria, microalbuminuria, neutrophil gelatinase-associatedlipocalin (NGAL), Cystatin C, interleukin 18 (IL-18), Kidney InjuryMolecule-1 (KIM-1), Liver Fatty Acid Binding Protein (L-FABP), LMP1,BARF1, IL-8, BRAF, CCNI, EGRF, FGF19, FRS2, GREB1, and LZTS1,alpha-amylase, carcinoembryonic antigen (CEA), CA 125, thioredoxin,beta-2 microglobulin levels—monitor activity of the virus, tumornecrosis factor-alpha receptors—monitor activity of the virus,Alpha-fetoprotein (AFP), CA15-3, CA 19-9, CYFRA 21-1, HE-4, PIVKA-11,ProGRP, SCC, follicle-stimulating hormone (FSH), leutinizing hormone(LH), T-cell lymphoma invasion and metastasis 1 (TIAM1), N-cadherin,EC39, amphiregulin, dUTPase, secretory gelsolin (pGSN), PSA (prostatespecific antigen), thymosin β15, insulin, plasma C-peptide, glycosylatedhemoglobin (HBA1c), C-Reactive Protein (CRP), Interleukin-6 (IL-6),ARHGDIB (Rho GDP-dissociation inhibitor 2), CFL1 (Cofilin-1), PFN1(profilin-1), GSTP1 (Glutathione S-transferase P), S100A11 (ProteinS100-A11), PRDX6 (Peroxiredoxin-6), HSPE1 (10 kDa heat shock protein,mitochondrial), LYZ (Lysozyme C precursor), GPI (Glucose-6-phosphateisomerase), HIST2H2AA (Histone H2A type 2-A), GAPDH(Glyceraldehyde-3-phosphate dehydrogenase), HSPG2 (Basementmembrane-specific heparan sulfate proteoglycan core protein precursor),LGALS3BP (Galectin-3-binding protein precursor), CTSD (Cathepsin Dprecursor), APOE (Apolipoprotein E precursor), IQGAP1 (RasGTPase-activating-like protein IQGAP1), CP (Ceruloplasmin precursor),and IGLC2 (IGLC1 protein), PCDGF/GP88, EGFR, HER2, MUC4, IGF-IR,p27(kip1), Akt, HER3, HER4, PTEN, PIK3CA, SHIP, Grb2, Gab2, PDK-1(3-phosphoinositide dependent protein kinase-1), TSC1, TSC2, mTOR, MIG-6(ERBB receptor feedback inhibitor 1), S6K, src, KRAS, MEKmitogen-activated protein kinase 1, cMYC, TOPO II topoisomerase (DNA) IIalpha 170 kDa, FRAP1, NRG1, ESR1, ESR2, PGR, CDKN1B, MAP2K1, NEDD4-1,FOXO3A, PPP1R1B, PXN, ELA2, CTNNB1, AR, EPHB2, KLF6, ANXA7, NKX3-1,PITX2, MKI67, PHLPP, adiponectin (ADIPOQ), fibrinogen alpha chain (FGA),leptin (LEP), advanced glycosylation end product-specific receptor (AGERaka RAGE), alpha-2-HS-glycoprotein (AHSG), angiogenin (ANG), CD14molecule (CD14), ferritin (FTH1), insulin-like growth factor bindingprotein 1 (IGFBP1), interleukin 2 receptor, alpha (IL2RA), vascular celladhesion molecule 1 (VCAM1) and Von Willebrand factor (VWF),myeloperoxidase (MPO), IL1α, TNFα, perinuclear anti-neutrophilcytoplasmic antibody (p-ANCA), lactoferrin, calprotectin, Wilm's Tumor-1protein, Aquaporin-1, MLL3, AMBP, VDAC1, E. coli enterotoxins(heat-labile exotoxin, heat-stable enterotoxin), influenza HA antigen,tetanus toxin, diphtheria toxin, botulinum toxins, Shiga toxin,Shiga-like toxin I, Shiga-like toxin II, Clostridium difficile toxins Aand B, etc.

Exemplary targets may be measured in a sample such as an environmentalsample, a biological sample obtained from a patient or subject in needusing the subject methods include: drugs of abuse (e.g. cocaine),protein biomarkers (including, but not limited to, Nucleolin, nuclearfactor-kB essential modulator (NEMO), CD-30, protein tyrosine kinase 7(PTK7), vascular endothelial growth factor (VEGF), MUC1 glycoform,immunoglobulin μ Heavy Chains (IGHM), Immunoglobulin E, αvβ3 integrin,α-thrombin, HIV gp120, NF-κB, E2F transcription factor, HER3,Plasminogen activator inhibitor, Tenascin C, CXCL12/SDF-1, prostatespecific membrane antigen (PSMA), gastric cancer cells, HGC-27; cells(including, but not limited to, non-small cell lung cancer (NSCLC),colorectal cancer cells, (DLD-1), H23 lung adenocarcinoma cells, Ramoscells, T-cell acute lymphoblastic leukemia (T-ALL) cells, CCRF-CEM,acute myeloid leukemia (AML) cells (HL60), small-cell lung cancer (SCLC)cells, NCIH69, human glioblastoma cells, U118-MG, PC-3 cells,HER-2-overexpressing human breast cancer cells, SK-BR-3, pancreaticcancer cell line (Mia-PaCa-2), and infectious agents (including, but notlimited to, Mycobacterium tuberculosis, Staphylococcus aureus, Shigelladysenteriae, Escherichia coli O157:H7, Campylobacter jejuni, Listeriamonocytogenes, Pseudomonas aeruginosa, Salmonella O8, and Salmonellaenteritidis).

Exemplary targets that may be measured in a sample obtained from apatient or subject in need using the subject methods include, but arenot limited to: HBV core capsid protein, CDK2, E2F transcription factor,Thymidylate synthase, Ras, EB1, and Receptor for Advanced Glycated Endproducts (RAGE).

5. SAMPLES

As used herein, “sample”, “test sample”, “biological sample” refer tofluid sample containing or suspected of containing an analyte ofinterest. The sample may be derived from any suitable source. In somecases, the sample may comprise a liquid, fluent particulate solid, orfluid suspension of solid particles. In some cases, the sample may beprocessed prior to the analysis described herein. For example, thesample may be separated or purified from its source prior to analysis;however, in certain embodiments, an unprocessed sample containing theanalyte may be assayed directly. The source of the analyte molecule maybe synthetic (e.g., produced in a laboratory), the environment (e.g.,air, soil, fluid samples, e.g., water supplies, etc.), an animal, e.g.,a mammal, a plant, or any combination thereof. In a particular example,the source of an analyte is a human bodily substance (e.g., bodilyfluid, blood, serum, plasma, urine, saliva, sweat, sputum, semen, mucus,lacrimal fluid, lymph fluid, amniotic fluid, interstitial fluid, lunglavage, cerebrospinal fluid, feces, tissue, organ, or the like). Tissuesmay include, but are not limited to skeletal muscle tissue, livertissue, lung tissue, kidney tissue, myocardial tissue, brain tissue,bone marrow, cervix tissue, skin, etc. The sample may be a liquid sampleor a liquid extract of a solid sample. In certain cases, the source ofthe sample may be an organ or tissue, such as a biopsy sample, which maybe solubilized by tissue disintegration/cell lysis.

A wide range of volumes of the fluid sample may be analyzed. In a fewexemplary embodiments, the sample volume may be about 0.5 nL, about 1nL, about 3 nL, about 0.01 μL, about 0.1 μL, about 1 μL, about 5 μL,about 10 μL, about 100 μL, about 1 mL, about 5 mL, about 10 mL, or thelike. In some cases, the volume of the fluid sample is between about0.01 μL and about 10 mL, between about 0.01 μL and about 1 mL, betweenabout 0.01 μL and about 100 μL, or between about 0.1 μL and about 10 μL.

In some cases, the fluid sample may be diluted prior to use in an assay.For example, in embodiments where the source of an analyte molecule is ahuman body fluid (e.g., blood, serum), the fluid may be diluted with anappropriate solvent (e.g., a buffer such as PBS buffer). A fluid samplemay be diluted about 1-fold, about 2-fold, about 3-fold, about 4-fold,about 5-fold, about 6-fold, about 10-fold, about 100-fold, or greater,prior to use.

In some cases, the sample may undergo pre-analytical processing.Pre-analytical processing may offer additional functionality such asnonspecific protein removal and/or effective yet cheaply implementablemixing functionality. General methods of pre-analytical processing mayinclude the use of electrokinetic trapping, AC electrokinetics, surfaceacoustic waves, isotachophoresis, dielectrophoresis, electrophoresis, orother pre-concentration techniques known in the art. In some cases, thefluid sample may be concentrated prior to use in an assay. For example,in embodiments where the source of an analyte molecule is a human bodyfluid (e.g., blood, serum), the fluid may be concentrated byprecipitation, evaporation, filtration, centrifugation, or a combinationthereof. A fluid sample may be concentrated about 1-fold, about 2-fold,about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 10-fold,about 100-fold, or greater, prior to use.

In certain embodiments, the analyte is not amplified (i.e., the copynumber of the analyte is not increased) prior to the measurement of theanalyte. For example, in cases where the analyte is DNA or RNA, theanalyte is not replicated to increase copy numbers of the analyte. Incertain cases, the analyte is a protein or a small molecule.

6. VARIATIONS ON METHODS

The disclosed methods of determining the presence or amount of analyteof interest present in a sample may be as described above. The methodsmay also be adapted in view of other methods for analyzing analytes.Examples of well-known variations include, but are not limited to,immunoassay, such as sandwich immunoassay (e.g., monoclonal-polyclonalsandwich immunoassays, including enzyme detection (enzyme immunoassay(EIA) or enzyme-linked immunosorbent assay (ELISA), competitiveinhibition immunoassay (e.g., forward and reverse), enzyme multipliedimmunoassay technique (EMIT), a competitive binding assay,bioluminescence resonance energy transfer (BRET), one-step antibodydetection assay, homogeneous assay, heterogeneous assay, capture on thefly assay, etc. In some instances, the descriptions below may overlapthe method described above; in others, the descriptions below mayprovide alternates.

(a) Immunoassay

The analyte of interest, and/or peptides or fragments thereof, may beanalyzed using an immunoassay. The presence or amount of analyte ofinterest can be determined using the herein-described antibodies anddetecting specific binding to analyte of interest. Any immunoassay maybe utilized. The immunoassay may be an enzyme-linked immunoassay(ELISA), a competitive inhibition assay, such as forward or reversecompetitive inhibition assays, or a competitive binding assay, forexample. In some embodiments, one signal generating compound or signalgenerating substrate is attached to the capture antibody and thedetection antibody. Alternately, a microparticle employed for capture,also can function for detection.

A homogeneous format may be used. For example, after the test sample isobtained from a subject, a mixture is prepared. The mixture contains thetest sample being assessed for analyte, a first specific bindingpartner, and a second specific binding partner. The order in which thetest sample, the first specific binding partner, and the second specificbinding partner are added to form the mixture is not critical. The testsample is simultaneously contacted with the first specific bindingpartner and the second specific binding partner. In some embodiments,the first specific binding partner and any analyte of interest containedin the test sample may form a first specific binding partner-analyte ofinterest-antigen complex and the second specific binding partner mayform a first specific binding partner-analyte of interest-secondspecific binding partner complex. In some embodiments, the secondspecific binding partner and any analyte of interest contained in thetest sample may form a second specific binding partner-analyte ofinterest-antigen complex and the first specific binding partner may forma first specific binding partner-analyte of interest-second specificbinding partner complex. Moreover, the second specific binding partneris labeled with or contains a detectable label as described herein.

A heterogeneous format may be used. For example, after the test sampleis obtained from a subject, a first mixture is prepared. The mixturecontains the test sample being assessed for analyte of interest and afirst specific binding partner, wherein the first specific bindingpartner and any analyte of interest contained in the test sample form afirst specific binding partner-analyte of interest complex. Preferably,the first specific binding partner is an anti-analyte of interestantibody or a fragment thereof. The order in which the test sample andthe first specific binding partner are added to form the mixture is notcritical. Preferably, the first specific binding partner is immobilizedon a solid support. The solid support used in the immunoassay (for thefirst specific binding partner and, optionally, the second specificbinding partner) can be any solid support known in the art, such as, butnot limited to, a magnetic particle, a bead a nanobead, a microbead, ananoparticle, a microparticle, a membrane, a scaffolding molecule, afilm, a filter paper, a disc, or a chip (e.g., a microfluidic chip). Inthose embodiments where the solid support is a bead, the bead may be amagnetic bead or a magnetic particle. Magnetic beads/particles may beferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic orferrofluidic. Exemplary ferromagnetic materials include Fe, Co, Ni, Gd,Dy, CrO₂, MnAs, MnBi, EuO, and NiO/Fe. Examples of ferrimagneticmaterials include NiFe₂O₄, CoFe₂O₄, Fe₃O₄ (or FeO.Fe₂O₃). Beads can havea solid core portion that is magnetic and is surrounded by one or morenon-magnetic layers. Alternately, the magnetic portion can be a layeraround a non-magnetic core. The solid support on which the firstspecific binding member is immobilized may be stored in dry form or in aliquid. The magnetic beads may be subjected to a magnetic field prior toor after contacting with the sample with a magnetic bead on which thefirst specific binding member is immobilized.

After the mixture containing the first specific binding partner-analyteof interest complex is formed, any unbound analyte of interest isremoved from the complex using any technique known in the art. Forexample, the unbound analyte of interest can be removed by washing.Desirably, however, the first specific binding partner is present inexcess of any analyte of interest present in the test sample, such thatall analyte of interest that is present in the test sample is bound bythe first specific binding partner.

After any unbound analyte of interest is removed, a second specificbinding partner is added to the mixture to form a first specific bindingpartner-analyte of interest-second specific binding partner complex. Thesecond specific binding partner is preferably an anti-analyte ofinterest antibody that binds to an epitope on analyte of interest thatdiffers from the epitope on analyte of interest bound by the firstspecific binding partner. Moreover, also preferably, the second specificbinding partner is labeled with or contains a signal generating compoundor signal generating substrate, as described above.

The use of immobilized antibodies or fragments thereof may beincorporated into the immunoassay. The antibodies may be immobilizedonto a variety of supports, such as magnetic or chromatographic matrixparticles, latex particles or modified surface latex particles, polymeror polymer film, plastic or plastic film, planar substrate, amicrofluidic surface, pieces of a solid substrate material, and thelike.

(b) Sandwich Immunoassay

The sandwich immunoassay measures the amount of antigen between twolayers of antibodies (i.e., a capture antibody (i.e., at least onecapture antibody) and a detection antibody (i.e. at least one detectionantibody). The capture antibody and the detection antibody bind todifferent epitopes on the antigen, e.g., analyte of interest. Desirably,binding of the capture antibody to an epitope does not interfere withbinding of the detection antibody to an epitope. Either monoclonal orpolyclonal antibodies may be used as the capture and detectionantibodies in the sandwich immunoassay.

Generally, at least two antibodies are employed to separate and quantifyanalyte of interest in a test sample. More specifically, the at leasttwo antibodies bind to certain epitopes of analyte of interest or ananalyte of interest fragment forming an immune complex which is referredto as a “sandwich”. One or more antibodies can be used to capture theanalyte of interest in the test sample (these antibodies are frequentlyreferred to as a “capture” antibody or “capture” antibodies), and one ormore antibodies with a signal generating compound or signal generatingsubstrate that also bind the analyte of interest (these antibodies arefrequently referred to as the “detection” antibody or “detection”antibodies) can be used to complete the sandwich. In a sandwich assay,the binding of an antibody to its epitope desirably is not diminished bythe binding of any other antibody in the assay to its respectiveepitope. In other words, antibodies are selected so that the one or morefirst antibodies brought into contact with a test sample suspected ofcontaining analyte of interest do not bind to all or part of an epitoperecognized by the second or subsequent antibodies, thereby interferingwith the ability of the one or more second detection antibodies to bindto the analyte of interest. The capture antibody described above is anexample of a capture molecule. The detection antibody described above isan example of a detection molecule.

In one embodiment, a test sample suspected of containing analyte ofinterest can be contacted with at least one capture antibody (orantibodies) and at least one detection antibodies either simultaneouslyor sequentially. In the sandwich assay format, a test sample suspectedof containing analyte of interest (membrane-associated analyte ofinterest, soluble analyte of interest, fragments of membrane-associatedanalyte of interest, fragments of soluble analyte of interest, variantsof analyte of interest (membrane-associated or soluble analyte ofinterest) or any combinations thereof) is first brought into contactwith the at least one capture antibody that specifically binds to aparticular epitope under conditions which allow the formation of anantibody-analyte of interest complex. If more than one capture antibodyis used, a multiple capture antibody-analyte of interest complex isformed. In a sandwich assay, the antibodies, preferably, the at leastone capture antibody, are used in molar excess amounts of the maximumamount of analyte of interest or the analyte of interest fragmentexpected in the test sample.

Optionally, prior to contacting the test sample with the at least onefirst capture antibody, the at least one capture antibody can be boundto a solid support which facilitates the separation the antibody-analyteof interest complex from the test sample. Any solid support known in theart can be used, including but not limited to, solid supports made outof polymeric materials in the form of planar substrates or beads, andthe like. The antibody (or antibodies) can be bound to the solid supportby adsorption, by covalent bonding using a chemical coupling agent or byother means known in the art, provided that such binding does notinterfere with the ability of the antibody to bind analyte of interestor analyte of interest fragment. Moreover, if necessary, the solidsupport can be derivatized to allow reactivity with various functionalgroups on the antibody. Such derivatization requires the use of certaincoupling agents such as, but not limited to, maleic anhydride,N-hydroxysuccinimide, azido, alkynyl, and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

After the test sample suspected of containing analyte of interest isbrought into contact with the at least one capture antibody, the testsample is incubated in order to allow for the formation of a captureantibody (or capture antibodies)-analyte of interest complex. Theincubation can be carried out at a pH of from about 4.5 to about 10.0,at a temperature of from about 2° C. to about 45° C., and for a periodfrom at least about one (1) minute to about eighteen (18) hours, fromabout 2-6 minutes, or from about 3-4 minutes.

After formation of the capture antibody (antibodies)-analyte of interestcomplex, the complex is then contacted with at least one detectionantibody (under conditions which allow for the formation of a captureantibody (antibodies)-analyte of interest-detection antibody(antibodies) complex). If the capture antibody-analyte of interestcomplex is contacted with more than one detection antibody, then acapture antibody (antibodies)-analyte of interest-detection antibody(antibodies) detection complex is formed. As with the capture antibody,when the at least one detection (and subsequent) antibody is broughtinto contact with the capture antibody-analyte of interest complex, aperiod of incubation under conditions similar to those described aboveis required for the formation of the capture antibody(antibodies)-analyte of interest-detection antibody (antibodies)complex. Preferably, at least one detection antibody contains a signalgenerating compound or signal generating substrate. The signalgenerating compound or signal generating substrate can be bound to theat least one detection antibody prior to, simultaneously with or afterthe formation of the capture antibody (antibodies)-analyte ofinterest-detection antibody (antibodies) complex.

The order in which the test sample and the specific binding partner(s)are added to form the mixture for assay is not critical. If the firstspecific binding partner is attached to the signal generating compoundor signal generating substrate, then signal generating compound orsignal generating substrate-attached first specific bindingpartner-analyte of interest complexes form. Alternatively, if a secondspecific binding partner is used and the second specific binding partneris attached to the signal generating compound or signal generatingsubstrate, then signal generating compound or signal generatingsubstrate-attached complexes of first specific binding partner-analyteof interest-second specific binding partner form. Any unbound specificbinding partner, whether labeled or unlabeled, can be removed from themixture using any technique known in the art, such as washing.

Next, signal, indicative of the presence of analyte of interest or afragment thereof is generated. Based on the parameters of the signalgenerated, the amount of analyte of interest in the sample can bequantified. Optionally, a standard curve can be generated using serialdilutions or solutions of known concentrations of analyte of interest bymass spectroscopy, gravimetric methods, and other techniques known inthe art.

(c) Forward Competitive Inhibition

In a forward competitive format, an aliquot of labeled analyte ofinterest of a known concentration is used to compete with analyte ofinterest in a test sample for binding to analyte of interest antibody.

In a forward competition assay, an immobilized specific binding partner(such as an antibody) can either be sequentially or simultaneouslycontacted with the test sample and a labeled analyte of interest,analyte of interest fragment or analyte of interest variant thereof. Theanalyte of interest peptide, analyte of interest fragment or analyte ofinterest variant can be attached with a signal generating compound orsignal generating substrate. In this assay, the antibody can beimmobilized on to a solid support. Alternatively, the antibody can becoupled to an antibody, such as an antispecies antibody, that has beenimmobilized on a solid support, such as a microparticle or planarsubstrate.

The labeled analyte of interest, the test sample and the antibody areincubated under conditions similar to those described above inconnection with the sandwich assay format. Two different species ofantibody-analyte of interest complexes may then be generated.Specifically, one of the antibody-analyte of interest complexesgenerated contains a signal generating compound or signal generatingsubstrate while the other antibody-analyte of interest complex does notcontain a signal generating compound or signal generating substrate. Theantibody-analyte of interest complex can be, but does not have to be,separated from the remainder of the test sample prior to quantificationof the detectable product or detectable label. Regardless of whether theantibody-analyte of interest complex is separated from the remainder ofthe test sample, the amount of detectable product or detectable label(e.g., detectable signal) in the antibody-analyte of interest complex isthen quantified. The concentration of analyte of interest (such asmembrane-associated analyte of interest, soluble analyte of interest,fragments of soluble analyte of interest, variants of analyte ofinterest (membrane-associated or soluble analyte of interest) or anycombinations thereof) in the test sample can then be determined, e.g.,as described above. If helpful, determination can be done by comparingthe quantity of detectable product or detectable label (e.g., detectablesignal) in the antibody-analyte of interest complex to a standard curve.The standard curve can be generated using serial dilutions of analyte ofinterest (such as membrane-associated analyte of interest, solubleanalyte of interest, fragments of soluble analyte of interest, variantsof analyte of interest (membrane-associated or soluble analyte ofinterest) or any combinations thereof) of known concentration, whereconcentration is determined by mass spectroscopy, gravimetrically and byother techniques known in the art.

Optionally, the antibody-analyte of interest complex can be separatedfrom the test sample by binding the antibody to a solid support, such asthe solid supports discussed above in connection with the sandwich assayformat, and then removing the remainder of the test sample from contactwith the solid support.

(d) Reverse Competition Assay

In a reverse competition assay, an immobilized analyte of interest caneither be sequentially or simultaneously contacted with a test sampleand at least one labeled antibody. The analyte of interest can be boundto a solid support, such as the solid supports discussed above inconnection with the sandwich assay format.

The immobilized analyte of interest, test sample and at least onelabeled antibody are incubated under conditions similar to thosedescribed above in connection with the sandwich assay format. Twodifferent species analyte of interest-antibody complexes are thengenerated. Specifically, one of the analyte of interest-antibodycomplexes generated is immobilized and contains a signal generatingcompound or signal generating substrate while the other analyte ofinterest-antibody complex is not immobilized and contains signalgenerating compound or signal generating substrate. The non-immobilizedanalyte of interest-antibody complex and the remainder of the testsample are removed from the presence of the immobilized analyte ofinterest-antibody complex through techniques known in the art, such aswashing. Once the non-immobilized analyte of interest antibody complexis removed, the amount of signal generating compound or signalgenerating substrate in the immobilized analyte of interest-antibodycomplex is then quantified. The concentration of analyte of interest inthe test sample can then be determined by comparing the quantity ofdetectable signal as described above. If helpful, this can be done withuse of a standard curve. The standard curve can be generated usingserial dilutions of analyte of interest or analyte of interest fragmentof known concentration, where concentration is determined by massspectroscopy, gravimetrically and by other techniques known in the art.

(e) One-Step Immunoassay or Capture on the Fly Assay

In a one-step immunoassay or capture on the fly assay, a solid substrateis pre-coated with an immobilization agent. The capture agent, theanalyte and the detection agent are added to the solid substratetogether, followed by a wash step prior to detection. The capture agentcan bind the analyte and comprises a ligand for an immobilization agent.The capture agent and the detection agents may be antibodies or anyother moiety capable of capture or detection as described herein orknown in the art. The ligand may comprise a peptide tag and animmobilization agent may comprise an anti-peptide tag antibody.Alternately, the ligand and the immobilization agent may be any pair ofagents capable of binding together so as to be employed for a capture onthe fly assay (e.g., specific binding pair, and others such as are knownin the art). More than one analyte may be measured. In some embodiments,the solid substrate may be coated with an antigen and the analyte to beanalyzed is an antibody.

In some embodiments, a solid support (such as a microparticle)pre-coated with an immobilization agent (such as biotin, streptavidin,etc.) and at least a first specific binding member and a second specificbinding member (which function as capture and detection reagents,respectively) are used. The first specific binding member comprises aligand for the immobilization agent (for example, if the immobilizationagent on the solid support is streptavidin, the ligand on the firstspecific binding member may be biotin) and also binds to the analyte ofinterest. The second specific binding member comprises a signalgenerating compound or signal generating substrate and binds to ananalyte of interest. The solid support and the first and second specificbinding members may be added to a test sample (either sequentially orsimultaneously). The ligand on the first specific binding member bindsto the immobilization agent on the solid support to form a solidsupport/first specific binding member complex. Any analyte of interestpresent in the sample binds to the solid support/first specific bindingmember complex to form a solid support/first specific bindingmember/analyte complex. The second specific binding member binds to thesolid support/first specific binding member/analyte complex and thesignal generating compounds or signal generating substrates detected. Anoptional wash step may be employed before the detection. In certainembodiments, in a one-step assay more than one analyte may be measured.In certain other embodiments, more than two specific binding members canbe employed. In certain other embodiments, multiple signal generatingcompounds or signal generating substrates can be added. In certain otherembodiments, multiple analytes of interest can be detected.

The use of a one step immunoassay or capture on the fly assay can bedone in a variety of formats as described herein, and known in the art.For example the format can be a sandwich assay such as described above,but alternately can be a competition assay, can employ a single specificbinding member, or use other variations such as are known.

Combination Assays (Co-Coating of Microparticles with Ag/Ab)

In a combination assay, a solid substrate, such as a microparticle isco-coated with an antigen and an antibody to capture an antibody and anantigen from a sample, respectively. The solid support may be co-coatedwith two or more different antigens to capture two or more differentantibodies from a sample. The solid support may be co-coated with two ormore different antibodies to capture two or more different antigens froma sample.

Additionally, the methods described herein may use blocking agents toprevent either specific or non-specific binding reactions (e.g., HAMAconcern) among assay compounds. Once the agent (and optionally, anycontrols) is immobilized on the support, the remaining binding sites ofthe agent may be blocked on the support. Any suitable blocking reagentknown to those of ordinary skill in the art may be used. For example,bovine serum albumin (“BSA”), phosphate buffered saline (“PBS”)solutions of casein in PBS, Tween 20™ (Sigma Chemical Company, St.Louis, Mo.), or other suitable surfactant, as well as other blockingreagents, may be employed.

As is apparent from the present disclosure, the methods disclosedherein, including variations, may be used for diagnosing a disease,disorder or condition in a subject suspected of having the disease,disorder, or condition. For example, the sample analysis may be usefulfor detecting a disease marker, such as, a cancer marker, a marker for acardiac condition, a toxin, a pathogen, such as, a virus, a bacteria, ora portion thereof. The methods also may be used for measuring analytepresent in a biological sample. The methods also may be used in bloodscreening assays to detect a target analyte. The blood screening assaysmay be used to screen a blood supply.

7. MULTIPLEXING

The methods may include one or more (or alternately two or more)specific binding members to detect one or more (or alternately two ormore) target analytes in the sample in a multiplexing assay. Each of theone or more (or alternately two or more) specific binding members bindsto a different target analyte and each specific binding member isconjugated to a different signal generating compound or signal generatedsubstrate. For example, a first specific binding member binds to a firsttarget analyte, a second specific binding member binds to a secondtarget analyte, a third specific binding member binds to a third targetanalyte, etc. and the first specific binding member is labeled with afirst signal generating compound or first signal generating substrate,the second specific binding member is labeled with a second signalgenerating compound or second signal generating substrate, the thirdspecific binding member is labeled with a third signal generatingcompound or a third signal generating substrate, etc. In someembodiments, the conditions of the sample can be changed at varioustimes during the assay, allowing detection of the first signalgenerating compound or first signal generating substrate, the secondsignal generating compound or second signal generating substrate, thethird signal generating compound or third signal generating substrate,etc., thereby detecting one or more (or alternately two or more) targetanalytes. In some embodiments, the one or more (or alternately two ormore) signal generating compounds or signal generating substrates aredetected simultaneously. In some embodiments, the one or more (oralternately two or more) signal generating compounds or signalgenerating substrates are detected consecutively. In some embodiments,the one or more (or alternately two or more) signal generating compoundsor signal generating substrates generates a different detectable signal,such as a different wavelength of fluorescence signal.

Alternatively, each of the one or more (or alternately two or more)specific binding members binds to a different target analyte and eachspecific binding member is conjugated to a different solid support, suchas a different fluorophore bead. For example, a first specific bindingmember binds to a first target analyte, a second specific binding memberbinds to a second target analyte, a third specific binding member bindsto a third target analyte, etc., the first specific binding member islabeled with a first signal generating compound or first signalgenerating substrate, the second specific binding member is labeled witha second signal generating compound or second signal generatingsubstrate, the third specific binding member is labeled with a thirdsignal generating compound or a third signal generating substrate, etc.,and the first specific binding member is immobilized on a first solidsupport, the second specific binding member is immobilized on a secondsolid support, the third specific binding member is immobilized on athird solid support, etc. In some embodiments, the one or more (oralternately two or more) signal generating compounds or signalgenerating substrates generates a different detectable signal, such as adifferent wavelength or fluorescence signal, and the different solidsupports is detected simultaneously or consecutively.

In some embodiments, a first specific binding member binds to a firsttarget analyte, a second specific binding member binds to a secondtarget analyte, a third specific binding member binds to a third targetanalyte, etc., the first specific binding member, the second specificbinding member, the third specific binding member, etc. are labeled witha signal generating compound or a signal generating substrate, and thefirst specific binding member is immobilized on a first solid support,the second specific binding member is immobilized on a second solidsupport, the third specific binding member is immobilized on a thirdsolid support, etc. In some embodiments, the signal generating compoundsor signal generating substrates generates a detectable signal, such as adifferent wavelength or fluorescence signal, and the different solidsupports is detected simultaneously or consecutively.

8. KITS

Also provided herein is a kit for use in performing the above-describedmethods. The kit may include instructions for analyzing the analyte withthe disclosed methods. Instructions included in the kit may be affixedto packaging material or may be included as a package insert. Theinstructions may be written or printed materials, but are not limited tosuch. Any medium capable of storing such instructions and communicatingthem to an end user is contemplated by this disclosure. Such mediainclude, but are not limited to, electronic storage media (e.g.,magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM),and the like. As used herein, “instructions” may include the address ofan internet site that provides the instructions.

Alternatively or additionally, the kit may comprise a calibrator orcontrol, e.g., purified, and optionally lyophilized analyte of interestor in liquid, gel or other forms, and/or at least one container (e.g.,tube, microtiter plates or strips) for use with the methods describedabove, and/or a buffer, such as an assay buffer or a wash buffer, eitherone of which can be provided as a concentrated solution. In someembodiments, the kit comprises all components, i.e., reagents,standards, buffers, diluents, etc., which are necessary to perform theassay. The instructions also can include instructions for generating astandard curve.

The kit may further comprise reference standards for quantifying theanalyte of interest. The reference standards may be employed toestablish standard curves for interpolation and/or extrapolation of theanalyte of interest concentrations. The kit may include referencestandards that vary in terms of concentration level. For example, thekit may include one or more reference standards with either a highconcentration level, a medium concentration level, or a lowconcentration level. In terms of ranges of concentrations for thereference standard, this can be optimized per the assay. Exemplaryconcentration ranges for the reference standards include but are notlimited to, for example: about 10 fg/mL, about 20 fg/mL, about 50 fg/mL,about 75 fg/mL, about 100 fg/mL, about 150 fg/mL, about 200 fg/mL, about250 fg/mL, about 500 fg/mL, about 750 fg/mL, about 1000 fg/mL, about 10pg/mL, about 20 pg/mL, about 50 pg/mL, about 75 pg/mL, about 100 pg/mL,about 150 pg/mL, about 200 pg/mL, about 250 pg/mL, about 500 pg/mL,about 750 pg/mL, about 1 ng/mL, about 5 ng/mL, about 10 ng/mL, about12.5 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 40ng/mL, about 45 ng/mL, about 50 ng/mL, about 55 ng/mL, about 60 ng/mL,about 75 ng/mL, about 80 ng/mL, about 85 ng/mL, about 90 ng/mL, about 95ng/mL, about 100 ng/mL, about 125 ng/mL, about 150 ng/mL, about 165ng/mL, about 175 ng/mL, about 200 ng/mL, about 225 ng/mL, about 250ng/mL, about 275 ng/mL, about 300 ng/mL, about 400 ng/mL, about 425ng/mL, about 450 ng/mL, about 465 ng/mL, about 475 ng/mL, about 500ng/mL, about 525 ng/mL, about 550 ng/mL, about 575 ng/mL, about 600ng/mL, about 700 ng/mL, about 725 ng/mL, about 750 ng/mL, about 765ng/mL, about 775 ng/mL, about 800 ng/mL, about 825 ng/mL, about 850ng/mL, about 875 ng/mL, about 900 ng/mL, about 925 ng/mL, about 950ng/mL, about 975 ng/mL, about 1000 ng/mL, about 2 μg/mL, about 3 μg/mL,about 4 μg/mL, about 5 μg/mL, about 6 μg/mL, about 7 μg/mL, about 8μg/mL, about 9 μg/mL, about 10 μg/mL, about 20 μg/mL, about 30 μg/mL,about 40 μg/mL, about 50 μg/mL, about 60 μg/mL, about 70 μg/mL, about 80μg/mL, about 90 μg/mL, about 100 μg/mL, about 200 μg/mL, about 300μg/mL, about 400 μg/mL, about 500 μg/mL, about 600 μg/mL, about 700μg/mL, about 800 μg/mL, about 900 μg/mL, about 1000 μg/mL, about 2000μg/mL, about 3000 μg/mL, about 4000 μg/mL, about 5000 μg/mL, about 6000μg/mL, about 7000 μg/mL, about 8000 μg/mL, about 9000 μg/mL, or about10000 μg/mL.

Any specific binding members, which are provided in the kit mayincorporate an at least one signal generating compound, one or moresignal generating substrates, or the like, or the kit can includereagents for labeling the specific binding members or reagents fordetecting the specific binding members and/or for labeling the analytesor reagents for detecting the analyte. If desired, the kit can containone or more different signal generating compounds and/or signalgenerating or substrates. The specific binding members, calibrators,and/or controls can be provided in separate containers or pre-dispensedinto an appropriate assay format.

The kit may include one or more specific binding members, for example,to detect one or more target analytes in the sample in a multiplexingassay. The number of different types of specific binding members in thekit may range widely depending on the intended use of the kit. Thenumber of specific binding members in the kit may range from 1 to about10, or higher. For example, the kit may include 1 to 10 specific bindingmembers, 1 to 9 specific binding members, 1 to 8 specific bindingmembers, 1 to 7 specific binding members, 1 to 6 specific bindingmembers, 1 to 5 specific binding members, 1 to 4 specific bindingmembers, 1 to 3 specific binding members, 1 to 2 specific bindingmembers, 2 to 10 specific binding members, 2 to 9 specific bindingmembers, 2 to 8 specific binding members, 2 to 7 specific bindingmembers, 2 to 6 specific binding members, 2 to 5 specific bindingmembers, 2 to 4 specific binding members, 3 to 10 specific bindingmembers, 3 to 9 specific binding members, 3 to 8 specific bindingmembers, 3 to 7 specific binding members, 3 to 6 specific bindingmembers, 3 to 5 specific binding members, 3 to 4 specific bindingmembers, 4 to 10 specific binding members, 4 to 9 specific bindingmembers, 4 to 8 specific binding members, 4 to 7 specific bindingmembers, 4 to 6 specific binding members, 5 to 10 specific bindingmembers, 5 to 9 specific binding members, 5 to 8 specific bindingmembers, 5 to 7 specific binding members, 5 to 6 specific bindingmembers, 6 to 10 specific binding members, 6 to 9 specific bindingmembers, 6 to 8 specific binding members, 6 to 7 specific bindingmembers, 7 to 10 specific binding members, 7 to 9 specific bindingmembers, 7 to 8 specific binding members, 8 to 10 specific bindingmembers, 8 to 9 specific binding members, or 9 to 10 specific bindingmembers. Each of the one or more specific binding members may bind to adifferent target analyte and each specific binding member may beassociated with a different signal generating compound and/or signalgenerating substrate. For example, the kit may include a first specificbinding member that binds to a first target analyte, a second specificbinding member that binds to a second target analyte, a third specificbinding member that binds to a third target analyte, etc. and the firstspecific binding member is associated with a first signal generatingcompound and/or first signal generating substrate, the second specificbinding member is associated with a second signal generating compoundand/or second signal generating substrate, the third specific bindingmember is associated with a third signal generating compound and/orthird signal generating substrate, etc. In addition to the one or morespecific binding members, the kits may further comprise one or moreadditional assay components, such as suitable buffer media, and thelike. The kits may also include a device for detecting and measuring thesignal generating compound and/or signal generating substrate, such asthose described supra. Finally, the kits may comprise instructions forusing the specific binding members in methods of analyte detectionaccording to the subject invention, where these instructions for use maybe present on the kit packaging and/or on a package insert.

Optionally, the kit includes quality control components (for example,sensitivity panels, calibrators, and positive controls). Preparation ofquality control reagents is well-known in the art and is described oninsert sheets for a variety of immunodiagnostic products. Sensitivitypanel members optionally are used to establish assay performancecharacteristics, and further optionally are useful indicators of theintegrity of the kit reagents, and the standardization of assays.

The kit can also optionally include other reagents required to conduct adiagnostic assay or facilitate quality control evaluations, such asbuffers, salts, enzymes, enzyme co-factors, substrates, detectionreagents, and the like. Other components, such as buffers and solutionsfor the isolation and/or treatment of a test sample (e.g., pretreatmentreagents), also can be included in the kit. The kit can additionallyinclude one or more other controls. One or more of the components of thekit can be lyophilized, in which case the kit can further comprisereagents suitable for the reconstitution of the lyophilized components.One or more of the components may be in liquid form.

The various components of the kit optionally are provided in suitablecontainers as necessary. The kit further can include containers forholding or storing a sample (e.g., a container for a urine, saliva,plasma, cerebrospinal fluid, or serum sample, or appropriate containerfor storing, transporting or processing tissue so as to create a tissueaspirate). Where appropriate, the kit optionally also can containreaction vessels, mixing vessels, and other components that facilitatethe preparation of reagents or the test sample. The kit can also includeone or more sample collection/acquisition instruments for assisting withobtaining a test sample, such as various blood collection/transferdevices such as microsampling devices, micro-needles, or other minimallyinvasive pain-free blood collection methods; blood collection tube(s);lancets; capillary blood collection tubes; other single fingertip-prickblood collection methods; buccal swabs, nasal/throat swabs; 16-gauge orother size needle, circular blade for punch biopsy (e.g., 1-8 mm, orother appropriate size), surgical knife or laser (e.g., particularlyhand-held), syringes, sterile container, or canula, for obtaining,storing or aspirating tissue samples; or the like. The kit can includeone or more instruments for assisting with joint aspiration, conebiopsies, punch biopsies, fine-needle aspiration biopsies, image-guidedpercutaneous needle aspiration biopsy, bronchoaveolar lavage, endoscopicbiopsies, and laproscopic biopsies.

If desired, the kit can contain a solid support, such as a magneticparticle, bead, membrane, scaffolding molecule, film, filter paper,disc, or chip.

If desired, the kit can further comprise one or more components, aloneor in further combination with instructions, for assaying the testsample for another analyte, which can be a biomarker, such as abiomarker of a disease state or disorder, such as infectious disease,cardiac disease, metabolic disease, thyroid disease, etc.

9. EXAMPLES Example 1

FIG. 4 illustrates a microchamber array for digital immunoassay and amechanism of fluorescent signal amplification by an enzymatic reaction.In this example, the bead diameter was ˜2.7 um, the well diameter/depthwas ˜4 um/˜4 um, the enzyme was alkaline phosphatase, the fluorogenicsubstrate was fluorescein diphosphate, and the signal amplification wasfor 90 min at RT.

To evaluate the usefulness of the digital ELISA immunoassay, a handheld(˜10×10×12 in cm) optical imaging system based on embodiments of theinvention was developed. In the demonstration, zero and 10 femto M ofantigen solution (recombinant HBsAg) was applied. The first light sourceapplied to the detection vessel resulted in the images (A) and (D),respectively, shown in FIG. 6. The second light source applied to thedetection vessel resulted in the images (B) and (E), respectively, shownin FIG. 6. The image analyzer combined the images (A) with (B) thatresulted in image (C) and combined the images (D) with (E) that resultedin image (F) as shown in FIG. 6. Images (C) and (F) illustrate detectedpositions of “only bead” and “bead-and-enzyme” plotted by gray and whitedots, respectively; the white dot is identified to an immune-complexsignal. The performance of the handheld detector showed goodperformance—capturing a 100,000 chamber-area in an image—on the digitalassay.

Example 2

Digital Immunoassay Optical Detection of HBsAg (Hepatitis B VirusSurface Antigen)

Anti-HBsAg mouse monoclonal antibodies (prepared in-house) were coatedonto 3 μm diameter paramagnetic microparticles (Agilent Technologies)surface with EDC (1-ethyl-3-3-Dimethylaminopropyl) to prepare captureantibodies. After the washing, the coated paramagnetic microparticleswere added into buffer solution including protein.

Anti HBsAg goat polyclonal antibodies (prepared in-house) wereconjugated with alkaline phosphatase (Abbott) using a standard chemicalreaction known in the art to prepare detection antibodies. Theconjugated antibodies were purified using a routine gel-filtrationmethod known in the art. The purified conjugated antibodies were dilutedinto buffer solution including protein.

50 μL of the anti-HBsAg antibody microparticle solution and 50 μL ofalkaline phosphatase conjugated anti-HBsAg antibody solution wereincubated with 75 μL of human serum (with or without HBsAg). Afterincubation for 18 minutes at 37 degrees C., the beads were washed withbuffer solutions, and mixed with MUP or FDP solution, then loaded into awell (6 mm diameter) that had a microwell array at the bottom(approximately 400,000 of 5 μm diameter wells (femtoliter chamber)). Aheavy fluorinated oil (such as, for example, FC-40) was added over thetop of the well and the aqueous phase and oil phase were changed. Theaqueous phase (top phase) was removed and the well was set with thehandheld optical imaging system described herein.

Images (pictures) taken with the optical system are shown in FIGS. 7-11.The microparticles were detected by scattering image detection. Thefemtoliter chamber which has antibody coatedmicroparticle-HBsAg-Alkaline phosphate conjugated antibody complex(HBsAg immune-complex) was detected by fluorescent image detection. Thedetected signals were analyzed using the Image J software to countnumber of microparticle and number of femtoliter chambers which hasHBsAg immune-complex.

Example 3

FIG. 4 illustrates a microchamber array for digital immunoassay and amechanism of fluorescent signal amplification by an enzymatic reaction.In this example, the bead diameter was ˜2.7 um and beads included amixture of Qdot625 (Ex/Em:blue/red)-coated and non-coated (the ratio wasapproximately 10%), the well diameter/depth was ˜4 um/˜4 um. All beadsand red fluorescence of Qdot-coated beads were visualized bylight-scattering and fluorescence imaging, respectively.

To evaluate the usefulness of fluorescence imaging for the digital ELISAimmunoassay, a handheld (˜10×10×12 in cm) optical imaging system basedon embodiments of the invention were utilized. In this example, thesingle emission filter was a 515 nm longpass type of filter (e.g.,Semrok515LP). In the demonstration, 10% of a binary mixture of the redfluorescent and non-fluorescent beads was applied.

The imaging scheme utilized demonstrated that the emission filter didnot need to be changed. The imaging scheme utilized a first autofocusstep using the first light source which included a green LED (525 nm)and then applied the first light source to the detection vessel forscattering and bead imaging. This resulted in the image (A) shown inFIG. 12. In the next step, the second light source, which included ablue LED (450 nm), was applied to the detection vessel to excite thesamples in the detection vessel. This resulted in the fluorescence image(B) shown in FIG. 12. Next, the chromatic aberration was refreshed toinclude a red LED (625 nm) as the first light source. An autofocus wasperformed with this refreshed first light source and then applied to thedetection vessel for scattering. This resulted in the image (C) shown inFIG. 12. The second light source (the blue LED) was applied to thedetection vessel to excite the samples, which resulted in image (D)shown in FIG. 12.

The image analyzer combined the images (A) with (D) that resulted inimage (E) as shown in FIG. 12. ImageJ software was used to merge theimages (A) and (D). Image (E) illustrates detected positions of“bead-and-fluorescent species” plotted by green and red dots.

Various features and advantages of the invention are set forth in thefollowing claims.

1.-53. (canceled)
 54. A method of detecting an analyte of interest in aliquid droplet, which method comprises: (a) providing a first liquiddroplet containing an analyte of interest and a second liquid dropletcontaining at least one solid support, wherein the solid supportcomprises a specific binding member that binds to the analyte ofinterest; (b) using energy to exert a force to manipulate the firstliquid droplet with the second liquid droplet to create a mixture; (c)moving all or at least a portion of the mixture to an array of wells;(d) adding at least one detectable label to the mixture before and/orafter step (c); and (e) detecting the analyte of interest in the wellsusing a compact digital assay apparatus comprising: (i) a detectionvessel; (ii) a light source configured to emit light toward thedetection vessel; (iii) a single filter positioned to receive and passthrough a portion of light reflected from a sample in the detectionvessel, that originated from the light source, and receive and passthrough a portion of a fluorescence output from a sample in thedetection vessel; and (iv) a detector configured to receive a portion ofthe reflected light and a portion of the fluorescence output that passesthrough the single filter.
 55. The method of claim 54, wherein the lightsource is a light-emitting diode.
 56. The method of claim 55, whereinthe light source includes a plurality of light-emitting diodes.
 57. Themethod of claim 54, wherein the light source is comprised of more thanone light source.
 58. The method of claim 54, wherein the light sourceis configured to change colors.
 59. The method of claim 58, wherein thelight source is configured to change between a blue color and a greencolor.
 60. The method of claim 59, wherein the green color light sourcereflects off a sample in the detection vessel for the detector togenerate optical data identifying whether a bead is present in thesample.
 61. The method of claim 59, wherein the blue color light sourceexcites a sample in the detection vessel for the detector to generateoptical data identifying whether an enzyme is present in the sample. 62.The method of claim 54, wherein the output is a fluorescence generatedafter excitation of a sample in the detection vessel by the lightsource.
 63. The method of claim 54, wherein the output is generated by achemical reaction of a sample in the detection vessel.
 64. A method ofdetecting an analyte of interest in a liquid droplet, which methodcomprises: (a) providing a first liquid droplet containing an analyte ofinterest and a second liquid droplet containing at least one solidsupport, wherein the solid support comprises a specific binding memberthat binds to the analyte of interest; (b) using energy to exert a forceto manipulate the first liquid droplet with the second liquid droplet tocreate a mixture; (c) moving all or at least a portion of the mixture toan array of wells; (d) adding at least one detectable label to themixture before and/or after step (c); and (e) detecting the analyte ofinterest in the wells using a compact digital assay apparatuscomprising: (i) a detection vessel; (ii) a first light source configuredto emit light toward the detection vessel and at an angle relative tothe detection vessel; (iii) a second light source configured to emitlight toward the detection vessel; (iv) a single filter positioned toreceive and pass through a portion of light reflected from a sample inthe detection vessel, that originated from the first light source, andreceive and pass through a portion of a fluorescence output from asample in the detection vessel; and (v) a detector configured to receivea portion of the reflected light and a portion of the fluorescence thatpasses through the single filter.
 65. The method of claim 64, whereinthe detection vessel comprises an axis extending therethrough atsubstantially 90 degrees.
 66. The method of claim 65, wherein the firstlight source is positioned at an angle relative to the axis.
 67. Themethod of claim 66, wherein the angle is between 0 degrees and 90degrees.
 68. The method of claim 66, wherein the angle is between 45degrees and 90 degrees.
 69. The method of claim 66, wherein the angle is80 degrees.
 70. The method of claim 65, wherein the second light sourceis configured to emit a beam of light that travels along the axis. 71.The method of claim 65, wherein the second light source is laterallyoffset relative to the axis, and wherein the second light source isconfigured to emit a beam of light that travels parallel to the axis.72. The method of claim 64, wherein the second light source includesmore than one light source.
 73. The method of claim 64, wherein thefirst light source is a light-emitting diode.
 74. The method of claim73, wherein the first light source includes a plurality oflight-emitting diodes.
 75. The method of claim 64, wherein the firstlight source is comprised of more than one light source.
 76. The methodof claim 64, wherein the first light source is a green light emittingdiode.
 77. The method of claim 64, wherein the second light source is ablue light emitting diode.
 78. The method of claim 64, wherein thedetector is configured to generate a first image based on light thatoriginated from the first light source, the first image identifyinglocation of a bead in a sample in the detection vessel.
 79. The methodof claim 78, wherein the detector is configured to generate a secondimage based on fluorescence output from the sample after activation bythe second light source, the second image detecting presence of a labelin the sample in the detection vessel.
 80. The method of claim 64,wherein the first light source includes a color, and further wherein,the color is based on the single filter.
 81. A method of detecting ananalyte of interest in a liquid droplet, which method comprises: (a)providing a first liquid droplet containing an analyte of interest and asecond liquid droplet containing at least one solid support, wherein thesolid support comprises a specific binding member that binds to theanalyte of interest; (b) using energy to exert a force to manipulate thefirst liquid droplet with the second liquid droplet to create a mixture;(c) moving all or at least a portion of the mixture to an array ofwells; (d) adding at least one detectable label to the mixture beforeand/or after step (c); and (e) detecting the analyte of interest in thewells using a compact digital assay apparatus comprising: (i) a samplearray having a plurality of detection vessels; (ii) a first light sourceconfigured to emit light toward the detection vessels at an anglerelative to the detection vessels to illuminate a sample in thedetection vessels; (iii) a second light source configured to emit lighttoward the detection vessels without using a mirror or other reflectiveobject, the second light source further configured to activate a samplein the detection vessels to emit a fluorescence output; (iv) a filterpositioned to receive and pass through a portion of light reflected fromthe detection vessels that originated from the first light source, andreceive and pass through a portion of the fluorescence output from thesample in the detection vessels; and (v) a detector configured toreceive the reflected light and the fluorescence output from the samplethat pass through the filter and to generate optical data identifyingwhich vessels contain a bead and which vessels contain a label.
 82. Themethod of claim 81, wherein the sample array comprises an axis extendingtherethrough at substantially 90 degrees.
 83. The method of claim 82,wherein the first light source is positioned at an angle relative to theaxis.
 84. The method of claim 83, wherein the angle is between 0 degreesand 90 degrees.
 85. The method of claim 83, wherein the angle is between45 degrees and 90 degrees.
 86. The method of claim 83, wherein the angleis 80 degrees.
 87. The method of claim 82, wherein the second lightsource is configured to emit a beam of light that travels along theaxis.
 88. The method of claim 82, wherein the second light source islaterally offset relative to the axis, and wherein the second lightsource is configured to emit a beam of light that travels parallel tothe axis.
 89. The method of claim 81, wherein the second light sourceincludes more than one light source.
 90. The method of claim 81, whereinthe first light source is a light-emitting diode.
 91. The method ofclaim 90, wherein the first light source includes a plurality oflight-emitting diodes.
 92. The method of claim 81, wherein the firstlight source is comprised of more than one light source.
 93. The methodof claim 81, wherein the first light source is a green light emittingdiode.
 94. The method of claim 81, wherein the second light source is ablue light emitting diode.
 95. The method of claim 81, wherein theoptical data is an image identifying a location of the bead, if present,in the detection vessels, and the label, if present, in the detectionvessels.
 96. The method of claim 81, wherein the first light sourceincludes a color, and further wherein, the color is based on the filter.97. A method of detecting an analyte of interest in a liquid droplet,which method comprises: (a) providing a first liquid droplet containingan analyte of interest and a second liquid droplet containing at leastone solid support, wherein the solid support comprises a specificbinding member that binds to the analyte of interest; (b) using energyto exert a force to manipulate the first liquid droplet with the secondliquid droplet to create a mixture; (c) moving all or at least a portionof the mixture to an array of wells; (d) adding at least one detectablelabel to the mixture before and/or after step (c); and (e) detecting theanalyte of interest using a compact digital assay apparatus comprising:(i) a sample array including a plurality of samples positioned in aplurality of nanowells; (ii) a light source configured to emit lighttoward the sample array at an angle relative to the sample array toilluminate a sample in the sample array, the light source having awavelength between 450 nm and 550 nm; (iii) a single filter positionedto receive and pass through a portion of light reflected from theplurality of samples in the sample array, that originated from the lightsource; and (iv) a detector configured to receive a portion of lightreflected from the sample.
 98. The method of claim 97, wherein the lightsource is a light-emitting diode.
 99. The method of claim 98, whereinthe light source comprises a plurality of light-emitting diodes. 100.The method of claim 97, wherein the light source includes more than onelight source.
 101. The method of claim 97, wherein the light source isconfigured to change colors.
 102. The method of claim 101, wherein thelight source is configured to change between a blue color and a greencolor.
 103. The method of claim 102, wherein the green color lightsource reflects off a sample in the sample array for the detector togenerate optical data identifying whether a bead is present in thesample.
 104. The method of claim 97, wherein the angle is between 0degrees and 90 degrees relative to an axis oriented perpendicular to thesample array.
 105. The method of claim 97, wherein the angle is between45 degrees and 90 degrees relative to an axis oriented perpendicular tothe sample array.
 106. The method of claim 97, wherein the angle is 80degrees relative to an axis oriented perpendicular to the sample array.